Sn-2 myristate-containing edible oil

ABSTRACT

A nutritional fat or oil-based composition for increasing HDL cholesterol, decreasing LDL cholesterol and decreasing the LDL/HDL cholesterol ratio in human plasma is described. The composition can advantageously include at least 1% by weight myristic acid esterified at the sn-2 position in triglyceride molecules, includes between 10% and 40% by weight linoleic acid, and further includes between 30% and 65% by weight oleic acid and between 15% and 40% by weight total saturated fatty acids. The ratio of sn-2 myristic acid to sn-2 palmitic acid is typically greater than 1:1 and the sum of weight percentages for saturated, monounsaturated and polyunsaturated fatty acids equals 100%. In desirable cases, the composition is substantially cholesterol-free.

RELATED APPLICATIONS

NOT APPLICABLE.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for increasingthe level of HDL cholesterol, reducing LDL cholesterol and the ratio ofLDL to HDL in human plasma by supplementing or replacing conventionaldietary fats with a particular class of dietary fats.

BACKGROUND OF THE INVENTION

The following discussion is provided solely to assist the understandingof the reader, and does not constitute an admission that any of theinformation discussed or references cited constitute prior art to thepresent invention.

Over the past 40 years clinical research has been reported studyingdietary fats and their role in modulating major species of plasmalipoproteins. A number of review articles have been written on thesubject of coronary heart disease, controlling plasma cholesterol levels(e.g., Steinberg et al. 1999; JAMA, 282(21): 2043-2050), andspecifically on the role of dietary fats in altering plasma lipoproteinlevels (e.g., Mensink et al. 2003; Am J Clin Nutr, 77:1146-1155). Otherresearch has studied changes in lipoprotein levels resulting fromdietary fats that are rich in various fatty acids. For example,Tholstrup et al. (1994; Am J Clin Nutr, 59:371-377) studied changes inlipoprotein levels resulting from diets rich in different saturatedfatty acids including stearic acid (provided by shea butter), palmiticacid (palm oil) and lauric and myristic acids (provided by palm kerneloil).

For over thirty years researchers have studied and compared differentfatty acids for their abilities to raise or lower overall cholesterollevels in human plasma. While there are divergent opinions on manyaspects of this subject, most nutritional experts agree that thesaturated class of fatty acids (herein abbreviated SFA) raises totalcholesterol levels (herein abbreviated TC levels), while polyunsaturatedfatty acids (herein abbreviated PUFA) lower them, and monounsaturatedfatty acids (MUFA), e.g., oleic acid, are more neutral in their effect.

As a point of clarification and to avoid confusion, fats that containmostly SFA are termed saturated fats (or SATS) while those fatscontaining mostly MUFA are termed monounsaturated fats (or MONOS), andthose fats containing mostly PUFA are termed polyunsaturated fats (orPOLYS). Beyond this simplistic view, it also understood that metabolismof individual fatty acid species within each class, can impact HDL andLDL cholesterol levels to different degrees.

A number of research studies have used regression analysis to suggestthat of the more common SFAs including lauric acid (C12:0), myristicacid (C14:0), palmitic acid (C16:0) and stearic acid (C18:0) found inmany edible fats and oils, myristic acid with 14 carbon atoms and zerosites of carbon-carbon unsaturation (C14:0) appears to be most potent inelevating total cholesterol (TC) levels in the plasma. Consistent withthese findings, some manufacturers of processed foods avoid the use ofhardening fats such as coconut oil or palm kernel oil that contain highlevels of myristic acid, in favor of palm stearin and regular palm oilthat are also hardening fats, but contain high levels of palmitic andstearic acids instead.

Thus, a recently produced commercial margarine known as Smart Balance®buttery spread (GFA Brands, Inc., Cresskill, N.J.) that combines thebeneficial LDL cholesterol-lowering properties of PUFA, e.g., found insoybean oil, with the beneficial oil hardening property and HDLcholesterol-raising property of SFA, incorporates palm oil rather thanpalm kernel oil to achieve the requisite hardened texture. Thismargarine and related healthful fat blends are based upon the work ofSundram et al., described in U.S. Pat. No. 5,578,334, U.S. Pat. No.5,843,497, U.S. Pat. No. 6,630,192 and U.S. Pat. No. 7,229,653incorporated herein in their entireties. Sundram et al. describe acholesterol-free blended fat composition that combines a polyunsaturatedfat (with linoleic acid providing between 15% and 40% by weight of thecomposition), and a cholesterol-free saturated fat (with one or more SFAfrom the group including lauric, myristic, and palmitic providingbetween 20% and 40% by weight of the composition). The effect of thesaturated fat, i.e., palm oil, in this margarine is to increase both HDLand LDL cholesterol while the effect of the polyunsaturated vegetableoil is to lower LDL cholesterol. The net effect of regularly consumingsuch a fat blend composition instead of a typical American dietary fatwas shown to be a modest increase in the HDL concentration and anincrease in the HDL/LDL concentration ratio in the blood.

With regard to the selection of palm oil as a saturated fat, in U.S.Pat. No. 5,578,334 it has been shown by Khosla and Hayes (Biochem.Biophys. Acta; 1991, 1083: 46-50) that the combination of lauric andmyristic acids found in palm kernel oil or coconut oil can produce alarger LDL pool and a poorer (lower) HDL/LDL ratio than palmitic andoleic acids. Similarly, Mensink (Am J Clin Nutr, 1993; 57 (suppl.)711S-714S) points out that myristic acid is more hypercholesterolemicthan palmitic acid. These and other studies have led to the conclusionthat dietary 12:0 and 14:0 fatty acids are worse than 16:0 and 18:0 interms of raising LDL, and it has been reassuring that palm oil ratherthan palm kernel oil is usually used as hardstock in margarines and inbaking and frying shortenings. Consistent with these findings, Sundramet al. in the above-cited series of U.S. patents indicate that palmiticacid (rather than lauric or myristic acid) is the preferred saturatedfatty acid to be included in the fat composition (see, for example,claims 11 and 12 in U.S. Pat. No. 7,229,653).

As briefly discussed above, there is a body of research in which SFAs ofdiffering chain length have been studied for their abilities to increaseHDL and LDL plasma cholesterol levels. More recently, some research hasbeen reported concerning the positional effect of fatty acids within thetriglyceride molecule. That is, the ability of a fatty acid to alterplasma cholesterol levels may depend upon which of the threeglyceryl-ester positions, i.e., the sn-1 and sn-3 (end positions), orthe sn-2 (middle position) it occupies. This positional effect can bedue to the difference in enzymatic cleavage and preferential degradationversus absorption of the fatty acid. For example, the pancreatic lipaseenzymes that cleave individual fatty acids from the glycerol backbone ofthe fat molecule selectively hydrolyze and remove the fatty acids at thesn-1 and sn-3 positions while leaving the sn-2 fatty acid attached tothe glycerol backbone to generate a sn-2 monoglyceride. The latter canbe absorbed into the intestinal cells and reformed as a triglyceride orphospholipids for transport in the bloodstream. Some of these moleculescan reach the liver where they may affect cholesterol and triglyceridemetabolism in varied and complex ways. It is well known that free fattyacids liberated from TG by the action of various lipases in the gut,peripheral blood vessels, or adipose tissue can be catabolized toprovide energy for the body, or may be used in the re-synthesis oftriglycerides.

For the benefit of the reader, the following is a brief summarydescribing fat digestion, transport and oxidation. Fatty acids areprincipally ingested as triglycerides, i.e., fats and oils, which cannotbe immediately absorbed by the intestine. Fats are broken down into freefatty acids plus monoglycerides by the pancreatic lipase enzyme thatcomplexes with a protein called colipase which is necessary for itsactivity. The complex can only function at a water-fat interface. Forenzymatic fat digestion to be efficient, it is essential that fattyacids and fats be emulsified by bile salts from the gall bladder. Fatsare absorbed as free fatty acids and 2-monoglycerides, but a smallfraction is absorbed as free glycerol and as diglycerides. Once acrossthe intestinal barrier, fatty acids can be reformed into triglyceridesor phospholipids and packaged into chylomicrons or liposomes, which arereleased into the lymphatic system and then into the blood. Fats areeither stored or oxidized for energy, and the liver acts as the majororgan for fatty acid metabolism and the processing of chylomicronremnants and liposomes into the various lipoproteins including VLDL andLDL. Fatty acids synthesized in the liver are converted to triglyceridesand transported to the blood as VLDL. In peripheral tissues, lipoproteinlipase converts part of the VLDL into LDL and free fatty acids, whichare taken up for metabolism. LDL is taken up via LDL receptors by liverand other tissues. This provides a mechanism for uptake of LDL by thecell, and for its breakdown into free fatty acids, cholesterol, andother components of LDL.

When blood sugar is low, the hormone, glucagon, signals adipocytes toactivate hormone-sensitive lipase to convert triglycerides into freefatty acids. While the fatty acids have very low solubility in the blood(typically about 1 μM), the most abundant protein in blood, serumalbumin, binds free fatty acids, increasing their effective solubilityto ˜1 mM, allowing fatty acid transport to organs such as muscle andliver for oxidation when blood sugar is low. Fatty acid catabolism orbreakdown that results in the release of energy involves three majorsteps including activation and transport into the mitochondria, betaoxidation, and electron transport. More specifically, fatty acids enterthe mitochondria primarily through carnitine-palmitoyl transferase I(CPT-I). It is believed that activity of this enzyme is the ratelimiting step in fatty acid oxidation. Once inside the mitochondrialmatrix, fatty acids undergo beta-oxidation. During this process, twocarbon molecules (acetyl-CoA) are repeatedly cleaved from the fattyacid. The acetyl-CoA can then enter the Krebs Cycle, producing highenergy NADH and FADH, that are subsequently used in the electrontransport chain to produce high energy ATP for cellular processes.

SUMMARY OF THE INVENTION

The present invention concerns fat compositions and related methods forincreasing HDL and/or reducing the LDL:HDL ratio in human serum and/orimproving (i.e., decreasing) fasting blood glucose levels. This isaccomplished by creating and using a fat composition which includes anappropriate balance of fatty acids. In particular advantageous fatcompositions, substantial but not excessive levels of total saturatedfatty acids, myristic acid and/or lauric acid are included with low ormoderate levels of linoleic acid together with a correspondingpercentage of monounsaturated fatty acids (generally oleic acid). Incertain further advantageous compositions, effective levels of sn-2myristate without excessive sn-2 palmitate are included, together withthe appropriate levels of linoleic acid, oleic acid, and total saturatedfatty acids.

Thus, a first aspect of the invention concerns an edible fat compositionwhich includes between 10% and 40% by weight of the polyunsaturatedfatty acid, linoleic acid (18:2), and between 15% and 50% by weightsaturated fatty acids in toto, with the remainder to make up 100 percentbeing monounsaturated fatty acids (generally oleic acid, e.g., from 10to 75%) and minor amounts of other polyunsaturated fatty acids. (Thatis, the sum of weight percentages for saturated, monounsaturated andpolyunsaturated fatty acids equals 100%.) Preferably the fat compositionincludes no more than 40% by weight myristic acid or a combination ofmyristic acid and lauric acid. Also preferably, the fat compositionincludes no more than 20% by weight palmitic acid (more preferably nomore than 10%). Further, preferably the fat composition includes no morethan 10% stearic acid (more preferably no more than 5%). In certaincases, the specified level of linoleic acid is replaced with acombination of at least 2, 3, or 4 polyunsaturated fatty acids taken inany combination selected from the group consisting of linoleic acid,alpha-linolenic acid, eicosapentaenoic acid (EPA), and docosahexaenoicacid (DHA), preferably such combination includes 3-7, 3-10, 3-14.9,3-20, 3-30, 3-38, 5-10, 5-12, 5-14.9, 5-20, 5-30, 5-38, 10-12, 10-14.9,10-20, 10-30, 10-38, 12-14.9, 12-20, 12-30, 12-38, 15-30, or 15-38%linoleic acid. Highly preferably the fat composition is substantiallycholesterol-free.

In certain embodiments, the fat composition includes 10-30, 10-25,10-20, 10-15, 15-40, 15-30, 15-25% linoleic acid, or less than 15%linoleic acid (e.g., 3-5, 3-7, 3-10, 3-12, 3-14.9, 5-7, 5-10, 5-12,5-14.9, 10-12, 10-14.9, or 12-14.9% linoleic acid) and/or thecomposition includes no more than 40, 35, 30, 25, or 20%, e.g., 10 to20, 10 to 30, 10 to 40, 15 to 20, 15 to 25, 15 to 30, 15 to 35, 15 to40, to 25, 20 to 30, 20 to 35, 20 to 40, 25 to 30, 25 to 35, or 25 to40% myristic acid.

In certain embodiment, specifically including those embodimentsspecified above, the fat composition includes no more than 50% saturatedfatty acids (e.g., from 15 to 50, 15 to 40, 15 to 30, 20 to 50, 20 to45, 20 to 40, 20 to 35, or 20 to 30% by weight saturated fatty acids).Also preferably, palmitic acid (16:0) constitutes no more than 20% oftotal fat, more preferably no more than 15%, and still more preferablyno more than 12, 10, 9, 8, 7, 6, or 5% of the total. Stearic acidpreferably constitutes no more than 10%, more preferably no more than 9,8, 7, 6, 5, 4, or 3% of the fat by weight. Substantially the remainderof the fatty acids in the fat composition are preferably oleic acid(18:1), and/or the linoleic acid may be replaced with a combination ofpolyunsaturated fatty acids as indicated above. Preferably when otherpolyunsaturated fatty acids are included, the linoleic acid is at least10% of the total fat, e.g., 10-14.9% by weight.

In certain embodiments, the edible fat composition includes at least 1%and preferably at least 2 or 3% by weight myristic acid esterified atthe sn-2 position in triglyceride molecules, preferably from 10% to 40%by weight linoleic acid (or other percentage as specified herein), from30% to 65% by weight oleic acid (or a percentage sufficient to total100% after accounting for the percentages of polyunsaturated andsaturated fatty acids), and from 15 to 50% (preferably 15% to 40%) byweight saturated fatty acids in toto, where the weight ratio of sn-2myristic acid to sn-2 palmitic acid is at least 1:1.

In particular embodiments, consistent ingestion of the edible fatcomposition (e.g., as part of a daily diet) increases HDL cholesterol,decreases LDL cholesterol, and/or decreases the LDL/HDL cholesterolratio in human plasma and/or decreases the fasting blood glucoseconcentration.

For some embodiments, the weight ratio of sn-2 myristic acid to sn-2palmitic acid is at least 1:1, 1.1:1, 1.2:1, 1.3:, 1.4:1, 1.5:1, 1.7:1,2:1, 2.2:1, 2.5:1, 3:1, 3.5:1, or 4:1 or is in a range defined by takingany two of the just-specified ratio values as endpoints of the range;the weight ratio of sn-2 myristic acid to sn-2 lauric acid is at least0.8:1, 0.9:1, 1:1, 1.1:1, 1.2:1, 1.3:, 1.4:1, 1.5:1, 1.7:1, 2:1, 2.2:1,or 2.5:1 or is in a range defined by taking any two of thejust-specified ratio values as endpoints of the range; the weight ratioof sn-2 myristic acid to (sn-2 palmitic acid plus sn-2 lauric acid) isat least 0.3:1, 0.4.1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.8:1, 1:1, 1.2:1,1.5:1, 1.7:1, or 2:1, or is in a range defined by taking any two of thejust-specified ratio values as endpoints of the range; the weight ratioof sn-2 myristic to sn-2 stearic acid is at least 1:1, 1.1:1, 1.2:1,1.3:, 1.4:1, 1.5:1, 1.7:1, 2:1, 2.2:1, or 2.5:1, 3:1, 3.5:1, or 4:1.

In certain embodiments, at least 20, 30, 40, 50, 60, or 70% of themyristic acid esterified at the sn-2 position in triglyceride moleculesis produced by chemical or enzymatic interesterification or both; thecomposition includes at least 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or25%, or in a range of 3 to 25%, 3 to 20%, 3 to 10%, 3 to 5%, 5 to 25%, 5to 20%, 5 to 15%, 5 to 10%, 5 to 8%, 8 to 20%, 8 to 17%, 8 to 16%, 8 to15%, 8 to 12%, 10, to 25%, 10 to 20%, or 10 to 15%, 12 to 20%, 12 to17%, 12 to 16%, or 12 to 15% by weight myristic acid esterified at thesn-2 position in triglyceride molecules; no more than 2, 3, 4, 5, 6, 7,8, 9, 10, or 12% by weight of the triglyceride molecules which includemyristic acid have three myristic acid residues; at least 20, 30, 40, or50% of the triglyceride molecules which include myristic acid have onlytwo myristic acid residues; at least 20, 30, 40, or 50% of thetriglyceride molecules which include myristic acid have only onemyristic acid residue; the sn-2 myristic acid is esterified in glyceridemolecules selected from the group consisting of triglycerides,diglycerides, monoglycerides and combinations thereof; at least 70, 80,90, 95, or 97% of the sn-2 myristic acid is esterified withintriglyceride molecules.

Also in certain embodiments, the ratio by weight of saturated fattyacids to linoleic acid in the edible fat composition is greater than0.5, 0.6, 0.7 0.8, 0.9, 1.0, 1.1, 1.2, 1.5, 1.7, 2.0, 2.5, or 3.0, or isin a range of 0.5 to 3.0, 0.5 to 2.0, 0.5 to 1.0, 1.0 to 3.0, 1.0 to2.0, or 2.0 to 3.0.

For some embodiments, at least 50, 60, 70, 80, or 90% of thetriglyceride molecules having an sn-2 myristic acid carry unsaturatedfatty acids esterified at either one or both of the sn-1 and sn-3glyceride positions, e.g., selected from the group consisting of oleicacid, linoleic acid, alpha-linolenic acid, EPA, DHA and combinationsthereof; at least 40, 50, 60, 70, 80, or 90% of the triglyceridemolecules which include sn-2 myristic acid also include myristic acidesterified at either the sn-1 or sn-3 glyceride position or both, or ateither the sn-1 or sn-3 position but not both.

The edible oils of the present invention can advantageously be used inpreparation of any of a variety of different products. Thus, a relatedaspect of the invention concerns a prepared food product which includesthe edible fat composition of the preceding aspect or an embodimentthereof.

In certain embodiments, the prepared food product is a cooking oil, anoil spread (e.g., a margarine), a shortening, a salad dressing; abarbecue or dipping sauce or other condiment, a baked good (e.g., bread,tortilla, pastry, cake, cookie, or pie), or a dairy product (e.g., amilk, yoghurt, or cheese); in certain embodiments, the present ediblefat composition is 2 to 10, 5 to 15, 10 to 30, 30 to 50, or 50 to 100%by weight of the prepared food product.

Another related aspect concerns a human diet or diet formulation, whichis intended for or has the effect of increasing the concentration of HDLcholesterol, decreasing the LDL cholesterol, and/or increasing theHDL/LDL concentration ratio in human plasma, and/or decreasing thefasting blood glucose concentration, in which a substantial amount,e.g., 10 to 100%, 10 to 90%, 10 to 80%, 10 and 75%, 10 to 50%, 20 to100%, 20 to 80%, 20 to 60%, 30 to 100%, 30 to 80%, 50 to 100%, or 50 to80% by weight of the daily dietary fat is provided by the edible fatcomposition of the first aspect or an embodiment thereof.

In particular embodiments, the human diet formulation is provided inliquid form or in packaged form, e.g., indicated for weight loss, fornutritional supplementation or replacement for elderly patients orpatients with compromised digestive systems, or for improvement of apatient's lipoprotein profile.

Likewise, the invention provides a method of aiding a person to increasethe concentration of HDL cholesterol, decrease the LDL cholesterol,and/or increase the HDL/LDL cholesterol ratio in their plasma, and/ordecrease the fasting blood glucose concentration. The method involvesproviding dietary fat composition according to the first aspect above.

In certain embodiments, the dietary fat composition is or includes astructurally modified triglyceride-based dietary fat composition, wherethe dietary fat composition includes at least 1% and preferably at least2 or 3% by weight myristic acid esterified at the sn-2 position intriglyceride molecules, between 10% and 40% by weight linoleic acid,between 30% and 65% by weight oleic acid, and between 15% and 40% byweight total saturated fatty acids, where the ratio of sn-2 myristicacid to sn-2 palmitic acid is greater than 1:1 and/or the ratio of sn-2myristic acid to sn-2 stearic acid is greater than 1:1, and the sum ofweight percentages for saturated, polyunsaturated and monounsaturatedfatty acids equals 100%. Preferably the fat composition is substantiallycholesterol-free

In particular embodiments, the edible oil composition is as describedfor the first aspect above or an embodiment thereof; the edible oilcomposition is provided at least in part or primarily in one or moreprepared foods or diets or diet formulations (e.g., liquid dietformulations) as specified for an aspect above or an embodiment thereof.

In certain embodiments, the person suffers from high LDL cholesteroland/or from low HDL/LDL cholesterol ratio in their plasma; the person isclinically obese.

Similarly, another related aspect concerns a method of increasing theconcentration of HDL cholesterol, decreasing the LDL cholesterol, and/orincreasing the HDL/LDL cholesterol ratio, and/or decreasing the fastingblood glucose concentration, in the plasma of a human subject. Themethod involves consistently ingesting a dietary fat composition of thefirst aspect above, e.g., a structurally modified triglyceride-baseddietary fat composition which includes at least 1% and preferably atleast 2 or 3% by weight myristic acid esterified at the sn-2 position intriglyceride molecules, between 10% and 40% by weight linoleic acid,between 30% and 65% by weight oleic acid, and between 15% and 40% byweight total saturated fatty acids, where the ratio of sn-2 myristicacid to sn-2 palmitic acid is greater than 1:1 and/or the ratio of sn-2myristic acid to sn-2 stearic acid is greater than 1:1, and the sum ofweight percentages for saturated, polyunsaturated and monounsaturatedfatty acids equals 100%. Preferably the fat composition is substantiallycholesterol-free.

In particular embodiments, the dietary fat composition is as specifiedas an edible oil composition for the first aspect above or an embodimentthereof.

A further aspect concerns a method of preparing an edible fatcomposition by blending an edible oil rich in sn-2 myristate with atleast one other edible oil, thereby forming a blended edible oil of thefirst aspect above.

In certain embodiments, the edible fat composition includes at least 1%and preferably at least 2 or 3% by weight myristic acid esterified atthe sn-2 position in triglyceride molecules, between 10% and 40% byweight linoleic acid, between 30% and 65% by weight oleic acid, andbetween 15% and 40% by weight total saturated fatty acids. The ratio ofsn-2 myristic acid to sn-2 palmitic acid is greater than 1:1, and thesum of weight percentages for saturated, polyunsaturated andmonounsaturated fatty acids equals 100%. Preferable the edible oil richin sn-2 myristate, the at least one other edible oil, and/or the ediblefat composition are substantially cholesterol-free.

In certain embodiments, the edible oil rich in sn-2 myristate is formedby a method that includes enzymatic or chemical interesterification,generally resulting in an increase in the sn-2 myristate level; theedible fat composition is as specified for the edible oil of the firstaspect above or an embodiment thereof.

In some embodiments, the fat composition is formed by blending a higholeic vegetable oil (such as high oleic sunflower oil or high oleicsoybean oil) with palm kernel oil or coconut oil.

Additional embodiments will be apparent from the Detailed Descriptionand from the claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A. Overview

The present invention focuses on subtle differences in the human body'slipoprotein regulatory response to dietary fats, which can includedietary fats that contain a number of different saturated fatty acidmolecules that are esterified either at the same or at differentglyceryl carbons (sn-1, sn-2 or sn-3 structural isomer locations) withinthe triglyceride molecule (and/or mono- or di-glyceride molecule).Because human clinical studies involving large numbers of subjects anddiffering diets are very costly and labor-intensive, and because thereare so many experimental variables of interest, there is a limitedamount of experimental data relating to the effect on lipoproteinmetabolism of dietary saturated fatty acids of differing molecularweights (i.e., 10, 12, 14, 16, and 18 carbons per fatty acid). There isalso minimal clinical research data in which differing sn-1, sn-2 andsn-3 triglyceride structural isomers of dietary triglycerides have beenconstructed and fed to human subjects over a period of time, in whichthese isomers contain fixed amounts of specified saturated fatty acidslocated at different glyceryl carbons.

Thus, this invention further concerns compositions and methods forselectively increasing the plasma level of HDL “good” cholesterol(HDL-C), decreasing the level of LDL “bad cholesterol (LDL-C), and/orreducing the LDL/HDL ratio, and/or reducing fasting blood glucose byconsuming a substantially cholesterol-free nutritional fat or oil-basedcomposition which contains an appropriate balance of linoleic acid,oleic acid, and saturated fatty acids. In some cases, the compositioncan advantageously contain an appropriate amount of sn-2 myristic acidbut with low sn-2 palmitate and sn-2 stearate.

A few investigators have recently proposed that myristic acid could beincorporated into the triglyceride structure at the sn-2 position (i.e.,middle glyceryl ester position) to improve the health quality of dietaryfats by causing an increase in the level of HDL cholesterol. Conversely,it is believed that the amount of this fatty acid can be minimized atthe outer sn-1 and sn-3 positions of the triglyceride. This is becauselipase enzymatic cleavage during fat digestion removes the outer fattyacids while leaving most of the sn-2 fatty acid intact on the glycerolbackbone to be absorbed into the bloodstream as a monoglyceride.Accordingly, a number of researchers have prepared dietary fats enrichedwith milkfat in which much of its 10% by weight myristic acid islocated, by nature, at the sn-2 position of the triglyceride molecule.However, milkfat also contains approximately 26% by weight palmitic acid(16 carbon saturated fatty acid) that is also preferentially located atthe sn-2 position. While sn-2 palmitic acid in breast milk may bebeneficial to newborns, its benefit to adults is questionable.

As an alternative to using milkfat as a source of sn-2 myristic acid,triglyceride structural isomers with sn-2 myristate can also be producedusing the free fatty acid of myristate in interesterification reactionswith natural and/or synthetic fats. Structural rearrangement of naturaland synthetic triglycerides that contain myristic acid at the sn-1 andsn3 positions can also be used to move some of the myristate to the sn-2position. For example, interesterification can be used to randomize thedistribution of myristic acid over the three positions of thetriglyceride molecule. Researchers have proposed that regular dietaryintake of fats containing sn-2 myristate may allow the human lipoproteinregulatory system to respond by beneficially increasing plasmaHDL-cholesterol levels. While some nutritional experiments have exploredthe use of different levels of sn-2 myristic acid in dietary fats,few'investigators have systematically adjusted the level of otherdietary fatty acids (saturated, monounsaturated and polyunsaturated) inconcert with sn-2 myristic acid so as to further improve the dietaryfat, and further increase the HDL cholesterol level and the HDL/LDLratio, and none have recognized the advantageous balance characterizingthe present invention.

B. Clinical/Nutritional Studies

Obstacles in carrying out dietary experiments include the requirementfor substantial amounts of synthetic dietary fats that contain specificfatty acids integrated into the fat molecule at specific positions, aswell as stringent control over the subjects' intake of dietary fats.Nevertheless, in one such study, Sanders et al. (Am J Clin Nutr 2003;77:777-782) provided two similar meals to 17 healthy adult males (mealsat least 1 week apart) containing 50 g of fat consisting of eitherunrandomized (normal) cocoa butter (abbreviated N-cbutter) or randomized(interesterified) cocoa butter (abbreviated IE-cbutter). These two fatswere produced from a single batch of cocoa butter, therefore providingthe same fatty acid composition but different structural isomersexhibiting different melting points (35° C. for N- and 50° C. forIE-cbutter). While almost all the palmitic and stearic saturated fattyacids in N-cbutter are located at the sn-1 and sn-3 positions, theinteresterification process randomized the locations of fatty acids suchthat 22% of the resulting triglyceride molecules contained palmitic acid(10%) and stearic acid (12%) at the sn-2 position, thereby increasingthe melting point of the fat. This nutritional study compared only theshort term changes (3 and 6 hour postprandial) in the plasma levels oflipoproteins and fats. Remarkably, in the 6 hour period following eachmeal there were no measurable changes in TC and HDL cholesterol levels,but within 3 hours following the meals the increments in plasmapalmitic, stearic and oleic acids (mmol/L) were as much as three-foldgreater for N-cbutter than for IE-cbutter. This observation suggeststhat enzymatic digestion and absorption work much more efficiently onnatural cocoa butter than interesterified cocoa butter with its highermelting point that is significantly above body temperature. The studyalso suggests that meaningful dietary-related changes in TC and HDLcholesterol levels cannot be expected after single meals. Indeed, fourweek dietary regimens are typically recommended for establishing steadystate lipoprotein levels in the plasma.

In another nutritional study examining the lipoprotein response to asaturated fatty acid located at different positions in the triglyceridemolecule, Nelson et al. (Am J Clin Nutr 1999; 70:62-69) studied fullterm infants that were fed formula from birth to 120 days, where theformula contained 25-27% palmitic acid (16:0) in which either 6% of the16:0 (standard fat formula) or 39% of the 16:0 (synthesized fat formula)was esterified at the sn-2 glyceryl carbon. The standard fat formula wasmade from a blend of natural fats including palm olein, soybean,sunflower and coconut whereas the synthesized fat formula (having afatty acid composition nearly identical to the standard fat) was termed“Betapol-2” (manufactured by Loders Croklaan, Inc., The Netherlands).Betapol-2 contained interesterified palm oil in which a considerableportion of the 16:0 had been moved to the sn-2 glyceryl carbon. Otherinfants were breast-fed, in which the breast milk contained 23% 16:0 ofwhich 81% by weight was esterified at the sn-2 position. Blood sampleswere obtained at 30 and 120 days, and plasma lipoproteins fractionatedand assayed. The triacylglycerol-rich lipoproteins/chylomicrons werefractionated by ultracentrifugation and the lipid again assayed forpercentage of 16:0 in the sn-2 position of the triglyceride fromstandard fat-fed, synthesized fat-fed and breast milk-fed infants.Accordingly, from the blood samples, 8.3%, 15.8% and 28.0% of the 16:0was recovered in the sn-2 position for standard fat formula,interesterified fat formula and breast milk respectively. The authorssuggest that about 50% of the original 16:0 fatty acid located at thesn-2 position is conserved through the process of sn-1, sn-3 pancreaticlipase hydrolysis, intestinal absorption of the sn-2 monoglyceride andtriglyceride reassembly. More interestingly, plasma cholesterol,triglycerides, fatty acids, as well as apoA-I and apo B were measured.Infants fed the interesterified Betapol-2 showed a significantly poorerlipoprotein profile with lower plasma HDL cholesterol and apo A-1 aswell as higher apo B levels associated with LDL cholesterol, whileinfants fed the natural standard formula showed similar totalcholesterol levels but beneficially higher HDL levels after 120 days(1.6 vs. 1.2 mmol/L) and higher apo A-1 levels (127 vs. 100 mg/L). Thisstudy raises potential concerns over the use of interesterified, ascompared with natural saturated fats.

An extensive current review by Karupaiah et al. (Nutrition andMetabolism; 2007, 4:16, entitled “Effects of stereospecific positioningof fatty acids in triacylglycerol structures in native and randomizedfats: a review of their nutritional implications” discusses a large bodyof research relating to nutrition and the modulation of lipoproteinmetabolism resulting from alterations in the positioning of fatty acidsin triglycerides, and is relevant in understanding the context of thepresent invention. It is incorporated herein by reference in itsentirety.

Applicant has found a small number of animal and human nutritionalstudies that have investigated changes in plasma HDL and LDL cholesterollevels when dietary fats are provided that contain at least a portion ofthe myristic acid (14:0) at the sn-2 glyceryl carbon of the triglyceridemolecule. For example, Temme et al. (J. Lipid Res. 1997; 38:1746-1754)describe a human clinical study with 60 subjects consuming test fats(40% of the dietary energy) for 6 weeks following a run-in period.During this run-in, the subjects consumed a diet enriched with a higholeic acid margarine containing 24% saturated fat (essentially free ofmyristate), 70% oleic acid and 6% linoleic acid. During the study, 63%of the dietary fat (25% of energy) was replaced by either the high oleicmargarine or a synthetic fat made by interesterifying 34% palm stearin,17% high oleic sunflower oil, 9% sunflower oil and 40% trimyristin toprovide a fat containing 64% saturated fatty acids, 26% monounsaturates(oleic acid) and only 9% polyunsaturates (linoleic acid). Accordingly,the myristic acid accounted for 40%×25% energy=10% energy, while thesn-2 myristic acid statistically accounted for ⅓ or 3.3% energy or⅓×40%×63%=8% of the dietary fat. With 34% palm stearin present alongwith the 40% trimyristin in the interesterification mixture, asubstantial proportion of the sn-2 fatty acids were palmitic acid alongwith myristic acid. Subjects' lipids and lipoprotein levels weremeasured, and showed an average net increase in TC (10.6%), HDL (8.6%)and LDL (14.7%) cholesterol. Unfortunately, both the LDL/TC cholesterolratio and the LDL/HDL ratio increased. The former increased from 0.608to 0.622 while the latter increased from 2.06 to 2.18.

In a separate human study by Dabadie et al. (J. Nutr. Biochem. 2005;16(6): 375-382) 25 healthy monks were fed two diets for 5 weeks eachrespectively providing 30% and 34% of the calories from fat, in which 8%and 11% of these calories came from saturated fatty acids with 0.6% and1.2% coming from myristic acid representing 2.5% and 3.5% by weight ofthe fat. PUFAs (as linoleic acid) accounted for 6.3% of the caloriesrepresenting approximately 20% by weight of the fat for both diets.While no increase in HDL was reported, other beneficial effects werenoted including a decrease in TC, LDL cholesterol and triglycerides, anda beneficial increase in the apo A-1/apo B ratio.

In another human study by Dabadie et al. (Br. J. Nutr. 2006; 96(2):283-289) 29 healthy monks were fed two diets (33% and 36% of the dietarycalories from fat) for 3 months in which myristic acid provided 1.2% and1.8% of calories, alpha-linolenic acid provided 0.9%, and linoleic acidprovided 4.5% of calories (approximately 14% by weight of the dietaryfat). In the baseline diet, myristic acid provided 1.2% of calories,alpha-linolenic acid provided 0.4% of calories, and linoleic acidprovided 5.5% of calories. Much of the myristic and alpha-linolenicacids were reported to be in the sn-2 triglyceride position. Diet 1(with 3.7% by weight myristic acid) produced a small decrease in TC,LDL-C, HDL-C, TG and the TC/HDL-C ratio, while diet 2 (with 4.7% byweight myristic acid) produced no decrease in TC or LDL-C, but a smalldecrease in TG and the TC/HDL-C ratio, and a small increase (6%) inHDL-C.

Both of the above studies by Dabadie et al. employed milkfat as thesource of sn-2 myristic acid. In the first study the weight ratio ofsaturated fat:monounsaturated fat:polyunsaturated fat (abbreviatedS:M:P) in the test fats was maintained at approximately 30%:45%:25%, andin the second study approximately 34%:41%:25% with linoleic acid (18:2)representing approximately 15%-20% of the fat. Unfortunately the 2%-5%by weight levels of myristic acid in these dietary fats are small whencompared to the 12%-17% by weight palmitic acid in these same fats, inwhich the palmitic acid, like the myristic acid, is preferentiallylocated at the sn-2 position in the triglycerides. Accordingly, it isdifficult to interpret what lipoprotein changes measured were beingcaused by which of the fatty acids located at the sn-2 position.

Similarly, in a hamster study by Loison et al. (Br. J. Nutr. 2002;87:199-210), as much as 2.4% of the total dietary energy for the rodentswas provided by myristic acid from milkfat and/or lard in which much ofthe myristic acid is located in the triglyceride sn-2 position. As theinvestigators increased the level of myristic acid stepwise from 2% to4% to 6.5% and to 8.5% of the dietary fat, the levels of monounsaturatedfatty acids (as oleic acid) and polyunsaturates (predominantly aslinoleic acid) were maintained approximately constant at 40-45% and9-10% by weight of the fat, respectively. Only the levels of saturatedfatty acids varied appreciably where, as the level of myristic acid wasincreased stepwise from 2% to 8.5% by weight, the level of stearic aciddecreased from 14% to approximately 6% by weight. In this hamster study,the authors demonstrated substantial increases in both the HDLcholesterol levels and the ratios of HDL to non-HDL cholesterol whenmyristic acid was partially replacing stearic and palmitic acids.Interestingly, the levels of the non-HDL cholesterol (representingLDL-C+VLDL-C) remained relatively constant as the dietary level ofmyristic acid level was increased.

While the above results are interesting, the fatty acid compositionsprovided by Loison et al. cannot be used for constructing the presentlyinvented fat compositions for several reasons. The dietary fats ofLoison et al. contain a variety of other saturated fatty acids locatedat the sn-2 position besides myristic acid, including palmitic andlauric acids, at levels that are significantly higher than myristicacid, and the investigators did not investigate the degree to whichthese negatively impacted the plasma cholesterol profile. Also, theirfats incorporated milkfat and lard, two animal fats containingsubstantial levels of cholesterol that would have negatively impactedthe plasma cholesterol profile. Furthermore, the investigators failed toconsider adjusting the level of polyunsaturated fatty acids, forexample, while maintaining a constant level of sn-2 myristate. Applicantbelieves that testing these important variables would have significantlyaffected their resulting lipoprotein profiles.

As discussed above, a number of studies suggest that sn-2 myristic acidmay alter the level of cholesterol-containing lipoproteins includingLDL, VLDL and HDL cholesterol. However, it is not clear whether only thesn-2 myristate level in a dietary fat is critical and what role othersaturated, monounsaturated and polyunsaturated fatty acids play inincreasing HDL-C and decreasing the LDL-C/HDL-C ratio. For example, in asomewhat different system, Sundram et al. in U.S. Pat. No. 5,578,334,U.S. Pat. No. 5,843,497, U.S. Pat. No. 6,630,192 and U.S. Pat. No.7,229,653 showed that linoleic acid (18:2)-containing fat could be addedto a saturated fat (palm oil) that contained high levels of palmiticacid located in the sn-1 and sn-3 positions of the triglyceridemolecules to beneficially decrease LDL-C. At the same time, the palmoil-related increase in HDL-C would persist if the level of linoleicacid was not excessive, i.e., if it remained within the range of 15% to40% by weight of the dietary fatty acids while the saturated fatty acidsremained within the range of 20% to 40%. However, the above-citedpatents of Sundram et al. do not consider saturated fatty acids at anystereoisomer position including the sn-2 position, and do not suggest orpredict what would happen if their cholesterol-free saturated fattyacids (typically provided by palm oil with sn-1 and sn-3 palmitic acid)are replaced by sn-2 myristic acid from interesterified fats. Indeed,there is no suggestion by Sundram et al. to replace palmitic acid in anyof their compositions with myristic acid.

Notwithstanding recent lipoprotein research involving myristic acid,Applicant has discovered an extensive body of much earlier clinical datapublished in 1970 by McGandy and Hegsted (Am J Clin Nutr, 23 (10),1288-1298, 1970) before the importance of HDL cholesterol was recognizedand measured. Within the context of the present invention, Applicant hasanalyzed these data using an equation that allows the calculation ofVLDL cholesterol, then HDL cholesterol and the ratio of LDL/HDLcholesterol. Surprising and unanticipated results beyond thoseoriginally described and understood by McGandy et al. and beyond thesubsequent understanding by others skilled in the field have emergedfrom the recalculation of their data, leading to a better understandingof the role of sn-2 saturated fatty acids and the role of other dietaryfatty acids in regulating the HDL cholesterol level and the ratio of HDLto LDL cholesterol.

C. Saturated Fatty Acid Selection and Triglyceride Location

As taught by Khosla and Hayes (Biochem. Biophys. Acta, 1083: 46-50,1991) and by Sundram et al. (see above), among the various saturatedfatty acids present in single fats and fat blends that include palm oil,coconut oil and/or palm kernel oil, it is palmitic acid that has beenpreferred over lauric and myristic acid (see, for example, claims 11 and12 in U.S. Pat. No. 7,229,653) for providing a favorable human plasmalipoprotein profile. In fact, palmitic acid is present at a level ofover 40% by weight in palm oil, and the selection of natural fats andoils according to the teaching of Sundram et al. to increase plasma HDLand the HDL/LDL ratio does not suggest placing myristic acid (14:0) atthe sn-2 position of triglyceride molecules. With the exception ofmilkfat, natural fats and oils that contain substantial levels ofmyristic acid carry the myristic acid either at the sn-1 position (palmkernel oil) or the sn-3 position (coconut oil) rather than at the sn-2position. Alternatively, if a modified fat such as an interesterifiedvegetable fat were prepared as briefly mentioned in Sundram et al.,these patents make no suggestion to interesterify a specific fat or fatsaccording to any particular procedure that would place myristic acid atthe sn-2 position of triglyceride molecules.

For a human dietary fat, it is intriguing to consider the possibilitythat the glyceryl ester location of a saturated fatty acid among thesn-1, sn-2 and sn-3 positions within the triglyceride molecule mayaffect the HDL and LDL levels in different ways and to differentextents, with differing health consequences. It is also intriguing toconsider the possibility that substituting one saturated fatty acid foranother saturated fatty acid of differing carbon chain length (such asC14 myristate for C16 palmitate) at any one of the three glyceryl esterlocations may also affect HDL and/or LDL levels in different ways and todifferent extents. There have been a number of research studies in whicha dietary fat rich in one saturated fatty acid has been replaced by adietary fat rich in another saturated fatty acid (e.g., replacingcoconut fat rich in lauric and myristic acids by palm oil rich inpalmitic acid).

However, in the context of the present invention, there have been only asmall number of human dietary studies in which HDL and LDL cholesterollevels have been monitored, comparing two (or more) similar dietary fatcompositions in which a defined level of one saturated fatty acid with adefined triglyceride structure is replaced by a second saturated fattyacid with a similar triglyceride structure. For example, a dietary studythat compares the lipoprotein profile of people consuming natural palmoil and then interesterified palm oil would be comparing dietscontaining the same fatty acids in differing triglyceride structures.Thus, oleic acid (18:1) that predominates at the sn-2 position innatural palm oil would be partially displaced during interesterificationby palmitic acid that originally predominates at the sn-1 and sn-3positions in natural palm oil.

However, in 1970 McGandy and Hegsted (Am J Clin Nutr, 23 (10),1288-1298, 1970) published an elegant clinical study with 18 humansubjects who were followed for 1.5 years, in which normal dietary fatswere replaced by semisynthetic triglycerides providing 38% of the totaldaily calories. Each fat-containing diet was fed to subjects for a 4week period in a random sequence of 30 dietary fat formulations. Thesesemisynthetic fats were produced by interesterifying 25% by weight ofindividual saturated fatty acids (in the form of series of trisaturatedtriglycerides C12 through C18) with 75% by weight natural vegetable oilslow in saturated fatty acids (either olive or safflower oil).

In some other fat-containing diets, 60% by weight C10 saturated fattyacid (as trisaturated C10 medium chain triglycerides, aka, “MCT oil”)was first interesterified with 40% by weight of the longer chainsaturated fatty acids (as trisaturated triglycerides C14 through C18) toform “60-40 interesterified C10-SFA” products. Subsequently, either 50%or 80% by weight of these “60-40 interesterified C10-SFA” products wereblended with the balance (i.e., either 50% or 20% by weight) ofvegetable oil (either olive or safflower oil).

Serum samples were obtained from the subjects on days 24 and 28 forassay of total cholesterol (abbreviated TC), beta-lipoproteincholesterol, lipid phosphorous, and triglyceride levels (abbreviatedTG). For each subject and each assay, an average value (based upon twosamples) was calculated. For each subject, changes in these averagevalues were calculated for each dietary fat, and then mean changes fromthe baseline “Average American Diet” (A.A. Diet) was calculated for thewhole group of subjects.

At the time of the 1970 publication by McGandy et al., whilebeta-lipoprotein was understood as “bad” cholesterol (now known asLDL-cholesterol), the concept of HDL as “good” cholesterol was unknownand only elucidated in 1974 by Mahley et al. (Circ. Res. 35:713-721,1974). The estimation of VLDL cholesterol from the Friedwald equationdescribed in 1972 was also unknown at the time. Applicant is unaware ofany attempts to reanalyze the data of McGandy et al. However, based uponthe hunch that these data held valuable but undetected information,Applicant calculated the values of VLDL and HDL cholesterol and theLDL/HDL ratios for all dietary groups of subjects using theseexceptionally controlled diets and the original data tables of McGandyet al. The calculations are based upon the following equation:HDL-C=TC−(LDL-C+VLDL-C) where VLDL=TG/5 (Friedwald estimate)

The new data estimates based on McGandy et al. are provided in Tables 1and 2. These data include VLDL-C, HDL-C and LDL/HDL cholesterol ratios.Together with knowledge of lipoprotein metabolism from other sources, itis believed that novel dietary fats can be assembled that will be moreeffective than those fats described by McGandy et al. in beneficiallyincreasing plasma HDL-C while decreasing the LDL-C/HDL-C ratio. It isalso anticipated that these novel fats will be more effective than thosepreviously described in Sundram et al. in U.S. Pat. No. 5,578,334, U.S.Pat. No. 5,843,497 and U.S. Pat. No. 6,630,192.

The Key to Tables 1 and 2 is as follows:

S:M:P represents the relative proportions of the total dietary energy(in kilocalories, with 38% of the energy provided by fat-abbreviated 38%en), provided by the different categories of fatty acids, saturates (S),monounsaturates (M) and polyunsaturates (P).

P/S represents the weight ratio of polyunsaturated to saturated fattyacids provided in the dietary fat; TC total cholesterol; LDL-C lowdensity lipoprotein associated cholesterol; VLDL very low densitylipoprotein associated cholesterol; HDL-C high density lipoproteinassociated cholesterol; LDL-C/HDL-C represents the weight ratio of LDL-Cto HDL-C.

MCT (10:0) refers to a medium chain triglyceride carrying threesaturated fatty acids, each ten carbons in length.

Interesterified MCT+14 (60:40) refers to a fat made by interesterifying60 parts by weight MCT(10:0) and 40 parts myristic acid (14:0).Similarly Interesterified MCT+16 and MCT+18 refer to the use of palmiticand stearic acids in place of myristic acid.

Interesterified Olive+12 (75:25) refers to a fat made byinteresterifying 75 parts by weight olive oil and 25 parts lauric acid(12:0). Similarly, Interesterified Olive+14 (75:25) refers to a fat madeby interesterifying 75 parts by weight olive oil and 25 parts myristicacid (14:0). Similarly, Interesterified Safflower+12 (75:25) refers to afat made by interesterifying 75 parts by weight safflower oil and 25parts lauric acid (12:0).

Blends (MCT+14): Safflower (80:20) refers to a fat blend made by mixing80% by weight of the interesterified MCT+14 product described above,with 20% by weight safflower oil.

By analogy, Blends (MCT+16): Olive (50:50) refers to a fat blend made bymixing 50% by weight of the interesterified MCT+16 product describedabove, with 50% by weight olive oil.

Results from Table 1 and Table 2.

In the uppermost panel of Table 1 it is apparent that as the P/S ratiofor edible dietary fats increased, the level of HDL increased and theratio of LDL to HDL cholesterol decreased for these 20 subjectsconsuming 38% of their calories from these fats. This lipoproteinimprovement is not surprising, considering safflower oil compared tobutter.

In the second panel of Table 1, it is apparent that 14:0 and 16:0 fattyacids rather than 18:0, when interesterified with MCT (10:0) producehealthier interesterified fat products based upon the higher HDL andlower LDL/HDL ratios for these subjects. The 14:0 interesterified fat isalso healthier than the original 100% MCT triglyceride based on thesesame criteria. Since sn-1 and sn-3 fatty acids are cleaved duringdigestion, it appears likely that the remaining sn-2 monoglyceride withits 14:0 is healthier than the sn-2 monoglyceride containing 10:0. Thisresult only became apparent with the calculation of HDL herein.

The third panel of Table 1 presents results in which the dietary fat is75% by weight olive oil interesterified with 25% by weight of varioussaturated fatty acids ranging from 12:0 to 18:0. Although Applicantbelieves that the proportion of polyunsaturated fatty acids (9% byweight) in the dietary fat is inadequate, it is interesting that theHDL-C level is highest for the 14:0 product, and the LDL/HDL ratio islowest. This result again suggests that sn-2 myristic acid has thegreatest potential in formulating a dietary fat that can provide maximumlevels of HDL-C and the best ratio of good to bad cholesterol.

The fourth panel results in Table 1 are much like the third panel exceptthat 75% by weight safflower oil containing a large proportion oflinoleic acid (68% by weight) was substituted for 75% olive oil in theinteresterification reaction to produce the dietary fat that containedapproximately 50% polyunsaturated fatty acids. As a result, most of theabsolute values of LDL-C and HDL-C are lower than the correspondingvalues with olive oil because high levels of linoleic acid can depressboth LDL-C and HDL-C levels. The LDL-C/HDL-C ratios are also somewhatcompromised (undesirably higher than those with olive oil) suggestingthat the 50% level of linoleic acid in the interesterified products isexcessive. This compares with the 9% level with olive oil (see above).From these data it is apparent that the level of linoleic acid in adietary fat that will maximize HDL-C while minimizing the ratio ofLDL-C/HDL-C lies somewhere between 10% and 50%, and probably closer to15%-20% by weight of the dietary fat. The unusually low LDL-C level forthe interesterified 18:0-safflower oil product (129 mg/dL) is alsoremarkable. This suggests an atypical response associated with high 18:0interesterification that awaits further investigation.

The data presented in Table 2 are more difficult to interpret. Panel 1repeats the data provided in Table 1 to provide a baseline cholesteroland lipoprotein response of subjects to interesterified trisaturatedtriglycerides [e.g., MCT+14 (60:40), representing 60 parts of 10:0interesterified with 40 parts of 14:0]. Panels 2 and 3 provide thecorresponding responses of subjects consuming these same interesterifiedtrisaturates but blended either 80:20 or 50:50 with safflower oil (68%18:2 linoleic acid) in Panel 2, and olive oil in Panel 3 (olive oil: 11%18:2 linoleic acid+70% monounsaturated oleic acid).

Comparing line by line of data in Panel 2 with that in Panel 1, it isclear that the addition of polyunsaturated vegetable oil to atrisaturated fat-rich diet decreases LDL-C and VLDL-C while increasingHDL-C. Consequently, the resulting LDL/HDL cholesterol ratios decreased.

It is difficult to see a comparable pattern when similarly comparing thedata in Panel 3 with Panel 1. It is evident that adding an oleicacid-rich vegetable oil (olive) to a diet rich in trisaturated fat doesnot offset the cholesterolemic properties or improve the lipoproteinprofile of the diet to the same degree as a polyunsaturated vegetableoil (safflower) i.e., MONOS do not work well by themselves against SATS.On the other hand, the 50:50 blends with olive oil show a substantialimprovement in lipoprotein profile over the blends containing only 20%olive oil. Applicant notes that the 50:50 blends contain 6% linoleicacid while the 80:20 blends contain only 2%. It is likely that thehigher 18:2 level is substantially more effective in reducing LDL andincreasing HDL cholesterol, implying that a critical mass of linoleicacid is necessary to make any calculated lipoprotein improvement areality.

In summary, the data in Tables 1 and 2 derived from McGandy et al. showboth original and newly calculated lipoprotein levels in human subjects,and show changes in these levels for subjects whose diets includechanges from one dietary fat to another, from highly saturated MCTs, toMONOS, to highly unsaturated POLYS (by moving from one line of data tothe next in Tables 1 and 2). Unlike any of the other prior art studies,these data are particularly valuable because they show lipoproteinchanges brought about by a change from one saturated fatty acid toanother in otherwise identical dietary fats, where the saturated fattyacid is also being esterified into the sn-2 position of triglyceridemolecules. McGandy et al. thought they were exclusively testing thesimple addition of specific SFAs into set amounts of SFA, MUFA or PUFA,and did not anticipate that their design would also encompass the issueof triglyceride molecular structure. By focusing on their uniquemodulation of individual saturated fatty acids, it is possible todefinitively conclude that myristic acid was the most effectivesaturated fatty acid in the sn-2 position for beneficially and maximallyincreasing HDL-C in most situations. At the same time, Applicant findsthat sn-2 myristic acid significantly reduces LDL-C, depending upon thereference diet that is being considered. For example, in panel 3 ofTable 1, the LDL-C value of 149 mg/dL for the interesterified dietaryfat “Olive+14” is identical to that for simple olive oil, but whencompared to the other interesterified fats in the same group (thatdiffer only in the saturated fatty acid chain length) was beneficiallyand significantly lower even as the associated HDL-C was remarkablyhigher.

The newly calculated lipoprotein data after McGandy et al. lead to aconclusion that differs from the prior art. Others have used milkfat indietary fat blends to provide sn-2 myristate and improve the lipoproteinprofile in human and other mammalian plasmas. However, it is nowapparent from the recalculated data of McGandy et al. that the“non-myristate” saturated fatty acids including lauric acid (12:0) andpalmitic acid (16:0) in the sn-2 position of triglyceride molecules(present in milkfat) reduce/compromise the increased HDL-C level andconversely increase the LDL-C/HDL-C ratio. Thus milkfat that containsnearly three times more palmitic acid than myristic acid in the sn-2position is the wrong choice for an ideal fat. Applicant believes thatfor an added dietary fat to be effective in improving human lipoproteinprofiles over a broad population, it should contain more sn-2 myristicacid than sn-2 palmitic acid (and also more sn-2 myristic acid than sn-2lauric acid). Moreover, milkfat is also problematic because it containsa substantial level of cholesterol (at least 0.25% by weight) thatboosts plasma LDL-C while diminishing HDL-C. Stripping milkfat of itscholesterol would be helpful in this regard, but that would not reducethe level of palmitic acid. On the other hand, considering the saturatedcommercial vegetable fats, none of these contain appreciable amounts ofmyristic acid in the sn-2 position. Those that contain significantlevels of myristic acid (coconut oil and palm kernel oil) also containlauric acid at levels nearly three-fold greater than myristic, and thelauric acid rather than the myristic acid is preferentially esterifiedat the sn-2 position. Therefore, in this case, isomeric randomization bysame fat-interesterification is not a good option either.

Considering the limited options with currently available technology,Applicant has concluded that interesterification that combines myristicacid and/or trimyristin on the one hand, and either oleic acid/trioleinor at least one oleic acid-rich vegetable oil (e.g., varieties ofcanola, soybean or sunflower oil with medium to high levels of oleicacid) on the other hand, is a viable option for producing sn-2myristate-containing triglycerides in which at least one of the threefatty acids should be unsaturated.

TABLE 1 Cholesterol Response in Humans to Fats of Different SaturationS:M:P (%) LDL-C VLDL-C HDL-C LDL-C/ Fat (38% en) P/S TC mg/dl mg/dlmg/dl mg/dl HDL-C Butter 24:13:02 0.08 254 196 16 43 4.6 A.A. Diet19:13:06 0.32 221 154 16 51 3.0 Olive 7:27:04 0.57 217 149 13 55 2.7Safflower 5:07:26 5.2 178 116 12 50 2.3 MCT (10:0) 34:03:01 0.03 214 14824 41 3.6 Interesterified MCT + 14 34:03:01 0.03 253 181 18 54 3.4(60:40) MCT + 16 34:03:01 0.03 267 193 19 55 3.5 (60:40) MCT + 1834:03:01 0.03 226 162 19 45 3.6 (60:40) Olive 7:27:04 0.57 217 149 13 552.7 Interesterified Olive + 12 15:19:04 0.24 230 157 12 61 2.6 (75:25)Olive + 14 15:20:04 0.25 234 150 17 67 2.2 (75:25) Olive + 16 14:20:040.26 233 165 13 55 3 (75:25) Olive + 18 14:21:04 0.26 233 160 16 58 2.8(75:25) Safflower 5:07:26 5.2 178 116 12 50 2.3 InteresterifiedSafflower + 12 13:06:19 1.5 192 135 11 46 2.9 (75:25) Safflower + 1413:06:19 1.5 201 132 16 53 2.5 (75:25) Safflower + 16 13:06:19 1.5 206149 11 46 3.3 (75:25) Safflower + 18 12:07:19 1.5 202 130 15 57 2.3(75:25)

TABLE 2 Cholesterol Response in Humans to Fats of Different SaturationS:M:P (%) TC LDL-C VLDL-C HDL-C LDL-C/ Fat (38% en) P/S mg/dl mg/dlmg/dl mg/dl HDL-C Interesterified MCT + 14 34:03:01 0.03 253 181 18 543.4 (60:40) MCT + 16 34:03:01 0.03 267 193 19 56 3.5 (60:40) MCT + 1834:03:01 0.03 226 162 19 45 3.6 (60:40) Blends Safflower:MCT + 14)(20:80) 28:04:06 0.21 252 175 15 62 2.8 (50:50) 20:05:13 0.68 207 147 1446 3.2 Safflower:(MCT + 16) (20:80) 28:04:06 0.22 242 159 14 69 2.3Safflower:(MCT + 18) (20:80) 28:04:06 0.22 235 170 19 46 3.7 (50:50)19:05:14 0.70 208 144 15 49 2.9 Blends Olive:(MCT + 14) (20:80) 29:08:020.06 252 186 15 51 3.6 (50:50) 21:15:03 0.13 234 159 15 60 2.6Olive:(MCT + 16) (20:80) 28:08:02 0.06 242 168 15 59 2.8 Olive:(MCT +18) (20:80) 28:08:02 0.06 240 177 19 44 4 (50:50) 20:15:03 0.13 227 14715 65 2.3

D. Advantageous Fat Compositions

Thus, in view of the discussion above, preferably in the fat compositionat least 3% by weight of the composition is sn-2 myristic acid, whilethe amounts of sn-2 palmitate and sn-2 laurate are minimized. Thecomposition also preferably includes between 10% and 40% by weightlinoleic acid (helping to lower LDL-C), between 15% and 40% by weighttotal saturated fatty acids, and between 30% and 65% by weight oleicacid. Oleic acid is considered an essentially “neutral” fatty acid thatdilutes the saturated and polyunsaturated fatty acids that are morebiologically active in raising and lowering lipoprotein levels. Theratio of sn-2 myristic acid to sn-2 palmitic acid, and the ratio of sn-2myristic acid to sn-2 lauric acid are both greater than 1:1. The sum ofweight percentages for saturated, monounsaturated and polyunsaturatedfatty acids equals 100%.

The rationale for utilizing the above triglyceride composition as acomponent or replacement for a significant portion of ones dietary fatis as follows:

-   -   1. There is an extensive body of clinical evidence that dietary        intervention with edible fats and oils that increase plasma        levels of HDL cholesterol, while decreasing the LDL-C, VLDL-C        levels, and the ratio of LDL-C to HDL-C all provide substantial        health benefits in terms of reducing the risk of coronary heart        disease and other health problems.    -   2. Applicant has found new evidence that intake of dietary fat        providing between approximately 3% and 20% of the total fat as        sn-2 myristic acid is beneficial, in spite of variable and        sometimes contradictory increases and decreases in HDL-C, LDL-C        and LDL/HDL ratios (e.g., see Dabadie et al. and Temme et al.)        with sn-2 myristate relative to control diets. It is        hypothesized these inconsistencies are caused by variable intake        of non-myristate sn-2 saturated fatty acids (i.e., sn-2        palmitate and laurate) as well as variable intake of        polyunsaturated fatty acids (18:2).    -   Applicant has calculated HDL-C data from early 1970 clinical        experiments of McGandy et al. when HDL “good” cholesterol was        unknown, in which essentially all dietary saturated fatty acid        intake was controlled. It has become evident herein, for the        first time (see Table 1 and 2 below), that with an otherwise        invariant fatty acid-containing diet, sn-2 myristate can more        predictably boost HDL-C and reduce the LDL-C/HDL-C ratio than        other saturated fatty acids at the sn-2 position, which        typically degrade or diminish these lipoprotein indices. The        sn-2 myristic acid effect on the lipoprotein profile appears        most relevant when many or most of the neighboring fatty acids        in the triglyceride molecules are oleic acid. These particular        results were obtained by McGandy et al., when 75 parts olive oil        were interesterified with 25 parts myristate. When compared with        an olive oil-rich diet alone, the diet with semi-synthetic        interesterified myristic acid-olive oil surprisingly did not        alter plasma LDL-C levels (149 versus 150 mg/dL) even though it        increased HDL-C from 55 to 67 mg/dL.    -   3. Together with the novel findings calculated from McGandy et        al., Applicant re-evaluated other animal and human clinical        data, and determined that trisaturated triglycerides, especially        tripalmitin and to a somewhat lesser degree trimyristin, are        cholesterolemic in elevating TC and LDL-C (Snook et al., Eur. J.        Clin. Nutr. 1999, 53: 597-605) and should be minimized in the        diet (also see Mukherjee et al. J. Atheroscler. Res. 1969,        10(1): 51-54). The waxy high melting point tristearin        triglyceride molecule appears to be an exception, in part        because it is poorly digested.    -   Accordingly, it has been deduced that the stoichiometric ratios        of fatty acids (including myristic acid/trimyristin) combined in        interesterification reactions should be chosen to minimize the        formation of trisaturated triglycerides. This can be        accomplished for example by including a sufficient amount of        oleic acid in the reaction to act as diluent molecules. It is        suggested that triglycerides with either one or two saturated        fatty acids (but not three) can be produced by        interesterification, and are suitable for increasing sn-2        myristic acid levels in dietary fats. Therefore, the        stoichiometric ratio of oleic acid (or ratio of oleic acid plus        linoleic acid) to myristic acid residues should approach 1:1,        and preferably somewhat greater than 1.0/1.0 to keep trimyristin        triglyceride concentrations below 10% by weight. For example, in        a 1:1 interesterification mixture of oleic (0) and myristic        acid (M) residues, approximately ⅛ of the molecules are        trimyristin (MMM) if the eight (sn-1-sn-2-sn-3) triglycerides        are randomly constituted as OMM, MOM, MMO, MOO, OMO, OOM, OOO,        and MMM.    -   4. The proportion of polyunsaturated fatty acids, i.e., linoleic        acid, to be used in a dietary fat composition is also an        important consideration. The concept of balanced fatty acids        between polyunsaturated and saturated fatty acids is described        by Sundram et al. in a series of U.S. patents cited above, with        the proportion of linoleic acid being set between 15% and 40% by        weight and the total saturates being set between 20% and 40% by        weight. Considering the information herein from the re-analysis        of McGandy et al., it is believed that the range of linoleic        acid in a dietary fat can be modestly expanded to between 10%        and 40% by weight. This is based upon the observation that        adding as little as 8% by weight linoleic acid (provided by 75%        by weight olive oil) to an interesterified fat that contained        25% by weight myristic acid, substantially increased HDL-C while        decreasing LDL-C, as compared to the components alone (see        “olive oil” and “MCT+14” in Table 1). It is suggested that 10%        to 20% linoleic acid is the preferred range of linoleic acid for        balancing between 15% and 40% by weight of total saturated fatty        acids in the dietary fat.

Surprisingly, and contrary to conventional nutritional teaching,indefinitely higher levels of polyunsaturates are not better. That is,when the level of polyunsaturated fatty acids is increased above thatlevel required for achieving “balance”, the lipoprotein profile becomesdegraded. This is evident in Table 1, comparing homologousinteresterified fats all made with 25% saturated fat and either 75%olive oil (8% final linoleic acid in panel 3) or alternatively 75%safflower oil (51% final linoleic acid in panel 4). It is apparent thatthree out of the four olive oil-containing interesterified fats providedsuperior lipoprotein profiles based on higher HDL-C and lowerLDL-C/HDL-C ratios than those with safflower oil.

The proportion of saturated fatty acids in the dietary fat is also amatter to be considered. Clearly, the level of myristic acid and theproportion of myristate residues in the bioactive sn-2 position of thetriglyceride molecule are important. But it is an open question as towhether the overall proportion of saturated fatty acids in the diet iscritical (excluding myristate). In the top panel of Table 1 withoutconsidering the butter diet, it is apparent that as the saturated fattyacids substantially increase (and the P/S ratio decreases 16-fold from5.2 to 0.57 to 0.32), the lipoprotein profile is only moderatelydegraded. That is, the LDL-C to HDL-C ratio increases from 2.3 to 2.7 to3.0 but the HDL-C levels remain substantially constant (50-55 mg/dL). Onthe other hand, with the butter diet, the HDL-C value drops dramatically(to 41 mg/dL) and LDL-C increases dramatically (to approximately 200mg/dL from 150 mg/dL), believed to be in large part because PUFA becomeslimiting.

These data also suggest that dietary cholesterol is a more substantialproblem than saturated fatty acids in degrading a healthy lipoproteinprofile. However, because saturated animal fats in meats and butter arewidely consumed and contain substantial levels of cholesterol, andbecause cholesterol-free saturated vegetable fats (e.g., palm oil) areless common in the American diet, there is a common misconception thatall saturated fat is harmful. In fact, in light of the HDL-C andLDL-C/HDL-C data presented in Table 1, it is suggested that the range oftotal saturated fatty acids in a dietary fat can safely vary between 15%and 40% by weight in the substantial absence of dietary cholesterol andin the presence of adequate PUFA.

-   -   5. The proportion of monounsaturated fatty acids, i.e., oleic        acid, in the dietary fat represents the remaining content of        essentially neutral fatty acids after considering the more        biologically active polyunsaturated and saturated fatty acids.        As explained above, if the proportion of linoleic acid is added        to oleic acid, then the calculated ratio of (oleic acid+linoleic        acid) to total saturated fatty acids in the interesterification        reaction should approach 1.0:1.0, and preferably exceed 1:1, so        as to minimize the formation of trisaturate triglycerides.        Accordingly, the dietary fat should contain between 30% and 65%        by weight oleic acid depending upon the level of saturated fatty        acids in the reaction.    -   6. The cholesterol concentration in a dietary fat should be        minimized to avoid degrading the lipoprotein profile (see        problematic butter, panel 1, Table 1). It is preferable that        dietary cholesterol not exceed 2 mg per serving as this is the        maximum permissible cholesterol level allowed under U.S. FDA        regulations for a product to be labeled as cholesterol-free. For        a 14 g serving of table spread, this level represents 0.014% by        weight cholesterol. By comparison, a low cholesterol food may        contain up to 20 mg (0.14% by weight) cholesterol per serving,        while butter typically contains 0.22% by weight cholesterol        (16-fold higher than a cholesterol-free product).

E. Interesterification Ratios

One objective of the present invention is to provide recipes for makinginteresterified fats that not only contain sn-2 myristic acid, but alsoprovide other fatty acids that will improve the human lipoproteinprofile. Considerations in such recipes not only include the choice ofingredients but also their concentrations that affect the yield ofvarious triglyceride products and the physical properties of theresulting fat such as melting point and crystallization tendency, e.g.beta prime versus beta crystals). Since interesterification involves arelatively random rearrangement process for fatty acids in thetriglyceride molecule, the practical percentage yield of sn-2 myristate,based upon input weight of two principal ingredients can vary overapproximately a two-fold range. Thus, a 3:1 mixture of trimyristate totrioleate will obviously produce a much greater yield (% by weight) oftriglycerides containing an sn-2 myristate than a 1:3 mixture of thesame materials. However, the melting point of triglycerides containing asubstantial proportion of trimyristate rather than dimyristate andmonomyristate is undesirably elevated. Consequently, Applicant favors astoichiometry in which one part of myristic acid and/or trimyristin isinteresterified with between 1 and 3 parts of an oleic acid-richvegetable oil (or alternatively oleic acid/triolein). McGandy et al.utilized a 75:25 or 3:1 ratio of vegetable oil to saturated fat insynthesizing the “olive+14” dietary fat in Table 1.

There are particular advantages in selecting other interesterificationratios, i.e., stoichiometries, in combining two or more different fatsand/or fatty acids. This is illustrated herein by example, when a 1:1molecular ratio of myristic acid to oleic acid is selected. Such a 1:1mixture in an interesterified fat can be constituted from trimyristintriglyceride and a high oleic acid vegetable oil such as sunflower oilcontaining 82% by weight oleic acid currently available from CargillInc. (Minneapolis, Minn.). The resulting triglyceride structuresproduced, for example, from 12 Myristic (M) fatty acids and 12 Oleic (0)fatty acids (where the fatty acids are randomly arranged along the sn-1,sn-2 and sn-3 positions to form 8 different stereoisomer structures, 7of which contain myristic acid) are as follows: MMM, MMO, MOM, OMM, MOO,OMO, OOM, OOO. Since two and three saturated fatty acids per moleculewill produce a “hard fat,” i.e., one that has a solid consistency atrefrigerator temperature, this interesterification producesapproximately 50% hard fat that can be very beneficial in producingmargarines and solid shortenings. By contrast, the 75:25 ratio of oliveoil and saturated fatty acid, e.g., myristic acid used in theinteresterifications described by McGandy et al. produces myristicacid-containing triglycerides that are almost exclusively monosaturated,i.e., OOM, OMO, MOO, and these triglycerides do not function to hardenvegetable oils.

The important contribution of McGandy et al. in the production andclinical study of the dietary fats including “olive+14” and“safflower+14” is recognized. However, based upon Applicant's currentre-analysis of their data, it is believed that a dietary fat must notonly contain at least 3% sn-2 myristate, but must also contain more(preferably at least two-fold more) sn-2 myristate than sn-2 palmitate,and more sn-2 myristate than sn-2 laurate to obtain the benefits of thepresent invention. This is because sn-2 palmitate and sn-2 laurateappear to negatively affect (increase) the LDL-C/HDL-C ratio (see“Olive+16” and “Safflower+16”). Applicant also finds that the dietaryfat compositions provided by McGandy et al. contain either too littlelinoleic acid (8% with 75:25 “olive+14”) or too much linoleic acid (51%with 75:25 “safflower+14”) for achieving the objectives describedherein, i.e., to maximize the level of HDL-C while minimizing theLDL-C/HDL-C ratio in human plasma. Accordingly, Applicant has increasedthe required level of linoleic acid above 8% by weight, to a level oflinoleic acid between 10% and 40% by weight of the dietary fat.Likewise, Applicant finds that some of the dietary fat compositions ofMcGandy et al. contain too little oleic acid and too much linoleic acid(14% 18:1 with 75:25 “safflower+14”) or plenty of oleic acid but toolittle linoleic acid (53% 18:1 with 75:25 “olive+14”) for achieving animproved lipoprotein profile. Therefore, a higher oleic acid level isused herein, in which the final level of oleic acid in the dietary fatcomposition is between 30% and 65% by weight so as to maximize the levelof HDL-C while minimizing the LDL-C/HDL-C ratio in human plasma.

The studies of Dabadie et al. and Loison et al. discussed earlier arealso acknowledged, but both of these groups utilized milkfat in theirstudies. With milkfat providing more sn-2 palmitic acid than sn-2myristic acid, their dietary fats are not compatible with the presentinvention and lipoprotein profiles cannot be optimized. Similarly, thedietary fats of Temme et al. discussed earlier that involvedinteresterifying high levels of palm stearin, myristin, and very lowlevels of linoleic acid are not compatible with the requirements of thepresent invention because substantial levels of sn-2 palmitin areproduced that offset the benefits of sn-2 myristin, and because thelevel of linoleic acid in the dietary fat is inadequate.

While the studies of McGandy et al. provide interesterified fats thatare somewhat closer to the requirements of the present invention, thelevels of polyunsaturated fatty acids required by the present inventionto reduce LDL cholesterol are not met. Furthermore, McGandy et al. doesnot explore what role interesterification stoichiometry plays inimproving the lipoprotein profile. That is, by varying the ratio ofoleic acid to myristic acid used for interesterification, it isanticipated that the lipoprotein profile can be altered. Morespecifically, McGandy et al. used a fixed 1:3 ratio of trisaturated fatto olive oil (70% oleic acid) or safflower oil (68% linoleic acid) toproduce interesterified triglycerides containing sn-2 saturated fattyacids (e.g., sn-2 myristate). This stoichiometry produces a predominanceof triglycerides containing a single saturated fatty acid. Applicantherein substantially varies this stoichiometric ratio, e.g., upward toapproximately 1:1, so as to introduce additional saturated fatty acidsinto the synthetic/semisynthetic dietary fat during interesterification.This has important consequences when interesterifying a trisaturatedtriglyceride or a simple saturated fatty acid with either amonounsaturated fatty acid-rich, or a polyunsaturated fatty acid-richfat. This change produces interesterified triglyceride moleculescontaining much more disaturated triglycerides. In the case of myristicacid interesterified with a high oleate or high linoleate vegetable oil,two out of the three disaturated triglycerides (with two myristates andone oleate or linoleate) will contain the bioactive sn-2 myristate.

The interesterified dimyristate triglycerides may also serve a secondfunction. As a hard fat, the disaturate triglycerides possess asignificantly higher melting point than the monosaturated triglyceridesformed by McGandy et al. (with two oleic or linoleic acids and onemyristate). The melting point however, remains well below thetemperature of the human mouth so that the fat has an excellent mouthfeel. This is particularly applicable for making margarine spreads andshortenings for example. Thus, depending upon how the lipoproteinprofile is affected by the presence of disaturates, the ratio ofmyristate to oleate and linoleate can be changed in theinteresterification reaction.

F. Fat Compositions Containing Low Levels of Linoleic Acid

Additional studies with experimental fat compositions were performedwith gerbils, using both natural oil blends and blends containinginteresterified oils. When considering these results it is important tonote that the gerbil is the most sensitive among all species, andprovides the best animal model for identifying the effect of dietaryfatty acids on the serum lipoprotein response (Pronczuk, A., P. Khosla,K. C. Hayes. Dietary myristic, palmitic, and linoleic acids modulatecholesterolemia in gerbils. FASEB J. 8:1191-1200, 1994.), especially forrevealing the importance of linoleic acid in lowering plasma cholesteroland LDL-C. The gerbil also appears to be useful for assessing the bloodglucose response to dietary fatty acids in a manner previously witnessedin humans. (Sundram K, Karupaiah T, Hayes K C. Stearic acid-richinteresterified fat and trans-rich fat raise the LDL/HDL ratio andplasma glucose relative to palm olein in humans. Nutr Metab 4:3, 2007.)In that human study, an IE fat made by interesterifying tri-18:0 withsoybean oil, was the cause of elevated blood glucose and a rise in theLDL/HDL ratio after only 1 month on diet.

In accordance with the results of these studies, the invention alsoincludes the additional discovery that in the proper balanced fat blend,levels of polyunsaturated fatty acids (especially linoleic acid) thatare lower than previously believed effective can induce an advantageousLDL/HDL ratio when ingested. In particular, the lower levels ofpolyunsaturated fatty acids are effective in decreasing the LDL/HDLratio in combination with suitable levels of monounsaturated fatty acids(generally oleic acid) and saturated fatty acids including sufficientbut not excessive levels of myristic acid or a combination of myristicacid and lauric acid. Much of the literature concerning fatty acidssuggests that the greater the level of polyunsaturated fatty acids inthe dietary fats, the better, because ingestion of unsaturated fattyacids, and in particular linoleic acid, is understood to decrease totalcholesterol in the blood. However, consumption of dietary fatscontaining high levels of linoleic acid decreases both LDL and HDL, butdoes not lead to the most beneficial reduction of the LDL/HDL ratio. Aspreviously described in U.S. Pat. Nos. 5,578,334; 5,843,497; 6,630,192;and 7,229,653, it was found by the present inventors that thecombination of between 15% and 40% by weight linoleic acid together withappropriate levels of saturated fatty acids (especially palmitic acid,16:0) and monounsaturated fatty acids (especially oleic acid, 18:1) isadvantageous to achieve a beneficial cholesterol lipoprotein ratio.

Thus, recent experiments by the present inventors indicate that thelevel and proportion of polyunsaturated fatty acids as linoleic acid(18:2) in the fat portion of the mammalian diet is important in alteringthe plasma LDL/HDL cholesterol ratio. Very surprisingly, an unexpectedlylow level of linoleic acid in the overall fat composition of the dietappears sufficient for decreasing LDL to nearly its lowest level, whenfed in the presence of a substantial but not excessive level of myristicacid (14:0) or the combination of lauric acid (12:0) and myristic acid(14:0) fatty acids. At the same time, this low level of linoleic acidappears important in allowing the beneficial HDL cholesterol level toremain high when fed with the 14:0 or the 12:0+14:0 combination. Theseresults indicate that of the 100% total of fatty acids (by weight)contained in a fat, less than 15% by weight (e.g., about 10-14.9% oreven as little as about 9, 8, 7, 6, 5, 4, or 3%) of linoleic acid can besufficient or even optimal when combined with a diet containing theappropriate levels of myristic or lauric-myristic fatty acid combinationto minimize the LDL/HDL cholesterol ratio.

As a result, even though the invention includes edible fat compositionswhich include 15 to 40% linoleic acid, surprisingly advantageous dietaryfat compositions (and food containing such fat compositions) can beprepared such that the fat composition contains less than 15% linoleicacid (e.g., 3-5, 3-7, 3-10, 3-12, 3-14.9, 5-7, 5-10, 5-12, 5-14.9,10-12, 10-14.9, or 12-14.9%). Such fat compositions also contain from 15to 50, 15 to 40, 15 to 30, 20 to 50, 20 to 45, 20 to 40, 20 to 35, or 20to 30% by weight saturated fatty acids. Myristic acid (14:0) preferablyprovides no more than 40% of the total fat by weight, more preferably nomore than 35, 30, 25, or 20%, e.g., 10 to 20, 10 to 30, 10 to 40, 15 to20, 15 to 25, 15 to 30, 15 to 35, 15 to 40, 20 to 25, 20 to 30, 20 to35, 20 to 40, 25 to 30, 25 to 35, or 25 to 40%. Also preferably,palmitic acid (16:0) constitutes no more than 20% of total fat, morepreferably no more than 15%, and still more preferably no more than 12,10, 9, 8, 7, 6, or 5% of the total fat. Stearic acid preferablyconstitutes no more than 10%, more preferably no more than 9, 8, 7, 6,5, 4, or 3% of the fatty acids by weight. Substantially the remainder ofthe fatty acids in the fat composition are preferably oleic acid (18:1)and can also include minor amounts of other polyunsaturated fatty acids.

As discussed above, advantageously the fat composition can includetriglycerides with a substantial percentage of myristic and/or lauricfatty acids esterified at the sn-2 position. Thus, preferably at least1% and preferably between 3% and 16% by weight is myristic acid and/orlauric acid located at the sn-2 position of the triglyceride molecule.Preferably the weight ratio of sn-2 myristic acid to sn-2 palmitic acidis greater than 1:1 and the sum of weight percentages for saturated,monounsaturated and polyunsaturated fatty acids equals 100%. In certaincases, the specified level of linoleic acid is replaced with acombination of at least 2, 3, or 4 polyunsaturated fatty acids taken inany combination selected from the group consisting of linoleic acid,alpha-linolenic acid, eicosapentaenoic acid (EPA), and docosahexaenoicacid (DHA). Highly preferably the fat composition is substantiallycholesterol-free.

Certain of the fat compositions can be prepared by blending differentfats having appropriate fatty acid profiles. For example, palm kerneloil or coconut oil can be used to provide substantial amounts ofmyristic and lauric acid. Based upon 100% by weight of the fatty acidscontained in a fat, palm kernel oil commonly contains about 49% lauricacid (12:0), about 17% myristic acid (14:0), about 8% palmitic acid(16:0), about 12% oleic acid (18:1), and about 2-3% linoleic acid(18:2), along with about 2-4% each of other saturated fatty acids(stearic 18:0, capric 10:0, and caprylic 8:0). Oleic acid can beprovided, for example, by blending in high oleic sunflower oil such asthat from Cargill Inc., Minneapolis or the high oleic soybean oil fromDuPont. The Cargill high oleic sunflower oil contains approximately 82%oleic acid, 8-9% linoleic acid and 8-9% saturated fatty acids, while theDuPont high oleic soybean oil contains approximately 84% oleic acid, 3%linoleic acid, and 13% saturated fatty acids. If desired, additionallinoleic acid can be contributed by adding any of a variety of vegetableoils containing substantial amounts of linoleic acid, e.g., standard orcommodity soybean, safflower, sunflower, and/or corn oils.

In addition to the blends of oils just indicated, blends can alsoinclude interesterified oils as described herein having increasedmyristic acid and/or (auric acid in the sn-2 position.

The tables below present gerbil study results for a range of test diets.

TABLE 3 Body and organ weights, blood glucose and plasma lipids ofgerbils fed control or IE fats for 4 weeks (GFA Gerbil 1) Diet #682 #683#684 #685 #686 AHA SFA tri14:0/HOSun tri16:0/HOSun tri18:0/HOSunINGREDIENT Control-1 Control-2 50/50 50/50 50/50 CHO:Fat:Protein (% en)41:41:18 41:41:18 41:41:18 41:41:18 41:41:18 Kcal/g 4.3 4.3 4.3 4.3 4.3Body weight (g) Initial   48 ± 2   48 ± 2   48 ± 2   48 ± 1   48 ± 1Final   67 ± 7   67 ± 5   66 ± 6   65 ± 5   66 ± 5 Food intake (g/d) 9.0 ± 0.6^(a)  9.6 ± 0.7^(b)  9.3 ± 0.9^(c)  9.4 ± 0.5^(d) 10.3 ±0.4^(a,b,c,d) (Kcal/d)   39 ± 3^(a)   41 ± 3^(b)   40 ± 4^(c)   40 ±2^(d)   44 ± 2^(a,b,c,d) Fast blood gluc, 4 wk (mg/dL)   85 ± 10   83 ±6   72 ± 8^(a,b)   97 ± 33^(a)  100 ± 15^(b) Small Intestine length (cm)34.4 ± 2.2^(a) 35.2 ± 2.1 33.9 ± 2.6^(b) 35.2 ± 0.8 36.9 ± 2.3^(a,b)Plasma TC (mg/dL)  131 ± 48^(a,b,c)  211 ± 65^(a)  175 ± 52  203 ±37^(b)  199 ± 54^(c) VLDL-C (mg/dL)   34 ± 5   44 ± 17   51 ± 29   52 ±14   68 ± 16 LDL-C (mg/dL)   25 ± 3^(a,b)   42 ± 8^(a)   33 ± 6^(c)   50± 11^(b,c)   37 ± 6 HDL-C (mg/dL)   68 ± 5^(a,b,c,d)  109 ± 13^(a)  104± 1^(b)  106 ± 10^(c)  102 ± 4^(d) LDL-C/HDL-C ratio 0.37 ± 0.04 0.38 ±0.03 0.32 ± 0.06^(a) 0.48 ± 0.15^(a) 0.36 ± 0.07 TG (mg/dL)  130 ± 152 112 ± 57   88 ± 68   72 ± 38  102 ± 84 Mean ± SD (n = 7-8, exceptlipoproteins obtained from combined 2-4 individual rat plasmas, n = 2-3)^(a,b,c,d)Means in a row sharing a common superscript are significanlydifferent (p < 0.05) using one-way ANOVA and Fisher's PLSD test

TABLE 4-1 Body and organ weights, blood glucose and plasma lipids ofgerbils fed control or experimental diets for 4 weeks #688 #687PKO/HOSun #689 #690 PKO 50/50 PKO/HOSun tri12:0/HOSun INGREDIENT Controlblend 60/40 IE 50/50 IE CHO:Fat:Protein (% en) 41:41:18 41:41:1841:41:18 41:41:18 Kcal/g   4.3   4.3   4.3   4.3 Body weight (g) Initial  50 ± 4   50 ± 4   50 ± 4   50 ± 4 Final   70 ± 4^(a)   67 ± 7   65 ± 7  69 ± 13^(b) Gain 0.62 ± 0.12^(a) 0.53 ± 0.18 0.48 ± 0.3 0.61 ±0.38^(b) (g/d) Food (g/d) 10.8 ± 0.8  9.9 ± 0.7 10.0 ± 0.8 10.5 ± 1.5intake (Kcal/d)   27 ± 2^(a)   25 ± 2^(a)   25 ± 2   26 ± 4 (Kcal/d/kgBW)  386 ± 29  373 ± 30  385 ± 31  377 ± 58 Fast Blood gluc, 4 wk   87 ±18   98 ± 21   82 ± 9^(a)  102 ± 21^(a,b,c,d) (mg/dL) Small Intestine  36 ± 2^(a)   35 ± 2   35 ± 2   36 ± 1^(b,c,d) length (cm) Plasma TC(mg/dL)  218 ± 52^(a,b,c,d,e)  170 ± 21^(a,f)  185 ± 20^(b)  191 ± 12VLDL-C (mg/dL)   31 + 10^(a)   42 ± 10^(b)   38 ± 5^(c)   25 ± 7^(d)LDL-C (mg/dL)   70 ± 10^(a,b,c,d,e,f,g,h)   35 ± 2^(a)   37 ± 3^(b)   50± 3^(c,h) HDL-C (mg/dL)  124 ± 25^(a,b,c,d,e,f)   98 ± 12^(a)  102 ±11^(b)  117 ± 4^(g) (HDL % of total) (57) (58) (55) (61) LDL-C/HDL-Cratio 0.58 ± 0.09^(a,,b,c,d) 0.36 ± 0.02^(a) 0.37 ± 0.06^(b) 0.42 ±0.03^(c) TG (mg/dL)   66 ± 33   42 ± 17   47 ± 24   69 ± 42

TABLE 4-2 Body and organ weights, blood glucose and plasma lipids ofgerbils fed control or experimental diets for 4 weeks (IE Study 2) #684#691 #692 #693 tri14:0/HOSun tri14:0/HOSun tri14:0/HOSun PKS/HOSunINGREDIENT 50/50 IE 40/60 IE 25/75 IE 60/40 IE CHO:Fat:Protein (% en)41:41:18 41:41:18 41:41:18 41:41:18 Kcal/g   4.3   4.3   4.3   4.3 Bodyweight (g) Initial   50 ± 4   50 ± 4   50 ± 4   51 ± 1 Final   65 ± 4  65 ± 5   68 ± 6   62 ± 2^(a,b) Gain 0.48 ± 0.16 0.47 ± 0.2 0.56 ±0.24^(c) 0.33 ± 0.08^(a,b,c) (g/d) Food (g/d) 10.3 ± 0.5 10.0 ± 0.7 10.4± 0.7 10.2 ± 0.5 intake (Kcal/d)   26 ± 1   25 ± 2   26 ± 2   26 ± 1(Kcal/d/kg BW)  400 ± 15  385 ± 31  382 ± 29  419 ± 16 Fast Blood gluc,4 wk   82 ± 10^(b)   93 ± 17   84 ± 17^(c)   85 ± 19^(d) (mg/dL) SmallIntestine   34 ± 2^(b)   34 ± 1^(a,c)   34 ± 1^(d) 33.6 ± 1.0^(b,f)length (cm) Plasma TC (mg/dL)  185 ± 23^(c)  178 ± 28^(d,g)  178 ±44^(e,h)  214 ± 37^(f.g.h) VLDL-C (mg/dL)   33 ± 4^(e)   39 ± 8^(f)   44± 17^(g)   69 ± 27^(a,b,c,d,e,f,g) LDL-C (mg/dL)   48 ± 12^(f,I)   40 ±7^(e.)   29 ± 10^(f,h,i,j)   47 ± 12^(g,,j) HDL-C (mg/dL)   97 ± 8^(c) 101 ± 8^(d,g)  103 ± 2^(e)  102 ± 0^(f) (HDL % of total) (52) (57) (58)(48) LDL-C/HDL-C ratio 0.49 ± 0.10^(f) 0.41 ± 0.10^(d) 0.28 ±0.10^(e,f,g) 0.47 ± 0.12^(g) TG (mg/dL)   54 ± 22   45 ± 13   61 ± 42  29 ± 12

TABLE 5 Fatty acid profile of experimental diets (Gerbils IE #1) Diet#686 #684 #685 tri18:0/ #682 #683 tri14:0/HOSun tri16:0/HOSun HOSun(PUFA AHA PO 50/50 50/50 50/50 % en) (13.5) (4.5) (3) (3) (3) Fatty acid% 8:0 + 10:0 0.0 0 0.0 0.0 0.0 12:0 0.0 0.3 0.0 0.0 0.0 14:0 0.6 1.145.8 0.0 0.2 16:0 28.2 42.9 2.0 42.7 1.7 18:0 7.2 4.6 1.8 3.0 44.1 18:133.5 39.3 42.0 44.6 43.7 18:2 29.0 10.7 7.7 7.8 7.6 18:3 3.5 0.4 0.0 0.00.0

TABLE 6 Fatty acid profile of experimental diets (gerbils IE s#2) Diet#688 #689 #690 #684 #691 #692 #693 #687 PKO/HOSun PKO/HOSuntri12:0/HOSun Tri14:0/HOSun tri14:0/HOSun tri14:0/HOSun PKS/HOSun (PUFAPKO 50/50 blend 60/40 IE 50/50 IE 50/50 IE 40/60 25/75 60/40 % en) (3)(3.5) (3) (3) (3) (3) (3) (3) Fatty acid % 8:0 + 10:0 5.6 2.9 3.4 0.00.0 0.0 0.0 2.3 12:0 44.5 22.7 26.3 44.6 0.0 0.0 0.0 28.2 14:0 15 7.79.3 0.2 45.8 36.5 21.3 12.0 16:0 8.8 6.3 6.7 1.8 2.0 2.2 2.6 6.7 18:02.3 3.8 3.0 1.8 1.8 2.1 2.8 3.2 18:1 16.6 47.6 44.2 42.7 42.0 51.0 64.540.2 18:2 7.2 8.7 7.0 7.6 7.7 7.2 7.1 7.4 18:3 0.0 0.0 0.0 0.0 0.0 0.00.0 0.0

Table 3 and Table 4 (Tables 4-1 and 4-2) above show the effects offeeding interesterified (IE) dietary fats to gerbils, including changesin plasma lipoproteins, triglycerides and blood glucose levels. Table 5lists the fatty acid profiles for the diets in the study correspondingto Table 3, while Table 6 lists the fatty acid profiles for the diets inthe study corresponding to Table 4.

Table 3 provides the results obtained from feeding five differentdietary fats to gerbils. These fats include a blend representing an AHA(American Heart Association) fat blend (control) providing a balance ofsaturated, monounsaturated, and polyunsaturated fatty acids inapproximately equal amounts (#682); a second control fat representing asaturated fat based on palm oil (#683); a third fat with tri-14:0(tri-myristic acid) interesterified with hi-oleic sunflower oil (HOSUN)at a 50/50 ratio (#684); a fourth fat similarly interesterified usingtri-16:0 (tri-palmitic acid) interesterified with hi-oleic sunflower oilat a 50/50 ratio; and finally a fifth fat similarly interesterifiedusing tri-18 (tri-stearic acid) interesterified with hi-oleic sunfloweroil at a 50/50 ratio.

Results in Table 3 show that all gerbils grew at the same rate, butthose fed tri-18:0 had to consume more food than all the other fatgroups in order to grow normally. This suggests that when stearic acidis interesterified into a normal oil at high concentration, theresulting IE fat is not metabolized efficiently, hampering growth. Thetri-18:0 fat also raised the blood glucose level relative to tri-14:0.In fact, the tri-14:0 IE fat induced the lowest fasting blood glucoselevel (72 mg/dL) and the lowest LDL-HDL ratio (0.32) among the fatstested. It was apparent that tri-14:0 produced the best metabolicresponse in terms of energy dynamics, as reflected in markers of fastingblood glucose and lipoprotein metabolism.

Table 4 extends the comparison of effects of different dietary fats ongerbil metabolism. The comparison now includes (palm kernel oil-PK0), anatural vegetable oil rich in both lauric acid (12:0) and myristic acid(14:0). Also included are natural vegetable oil blends and IE fatproducts made by combining PKO with HOSUN. This experiment was intendedto compare and further elucidate the functional efficacy ofinteresterifying vegetable oils as compared to the simple blending oftwo natural oils to achieve the desired fatty acid compositioncharacteristics. In addition, myristic acid (tri-14:0) and HOSUN oilwere combined at different ratios using interesterification. Finally,lauric acid (tri-12:0) was interesterified with HOSUN oil using a 50/50ratio of the oils.

As in Table 3, different IE fat compositions influenced blood glucoselevels to different extents. An elevated fasting glucose level (102mg/dL) was measured with dietary fat in which tri-12:0 wasinteresterifed 50/50 with HOSUN (diet #690). By comparison, the IE fatproduced from tri-14:0 and HOSUN using a 50/50 ratio again improved thefasting blood glucose level (82 mg/dL, diet #684), as did the IE fatproduced using a 60/40 ratio of PKO and HOSUN (82 mg/dL, diet #689). TheIE fat produced by interesterifying tri-14:0 and HOSUN, but using areduced ratio of 25/75 for tri-14:0 and HOSUN (diet #692) resulted in aparticularly favorable ratio of LDL/HDL cholesterol (0.28) as well as afavorable fasting glucose level of 84 mg/dL). In the same series ofexperiments, it is interesting to observe that natural PKO alone (diet#687) produced the worst ratio of LDL/HDL cholesterol. Collectively,these data suggest that interesterification that combines myristic acid(tri-14:0) and oleic acid (e.g., HOSUN oil), or the blending of PKO(providing both myristic and lauric acids (14:0 and 12:0) together witha high oleic acid-containing oil (e.g., HOSUN) can be particularlyadvantageous.

G. Definitions

In the context of the present invention and the associated claims, thefollowing terms have the following meanings:

The term “nutritional fat” or “dietary fat” as used herein means anypredominantly triglyceride-based edible oil or fat, regardless ofwhether it is derived or purified from vegetable or animal sources, oris synthetic or semi-synthetic in origin, or some combination of these.A nutritional or dietary fat may also contain other constituents ofchoice such as monoglycerides, diglycerides, flavorings, fat-solublevitamins, phytosterols and other edible ingredients, food additives,dietary supplements and the like. As taught in the present invention,certain of the dietary fat or oil-based composition can be formulated bychemically or genetically engineering a fat or oil using chemical orenzymatic interesterification to attach certain fatty acids (or removecertain fatty acids and attach others) to the glyceryl backbone of thefat. A nutritional or dietary fat can be interesterified by chemicaland/or enzymatic methods known in the art using defined ratios ofcarefully controlled ingredients to produce certain predictedtriglyceride products as taught herein.

The objective of the present invention is to increase HDL “good”cholesterol, decrease LDL “bad” cholesterol, and/or decrease the ratioof LDL to HDL cholesterol ratio in human plasma. Another effect can beto reduce fasting blood glucose levels.

It is important that the resulting fat-based composition issubstantially cholesterol-free because the presence of cholesteroldegrades the lipoprotein profile, undesirably increasing LDL cholesteroland increasing the LDL/HDL ratio in the plasma. The term “substantiallyfree” in reference to cholesterol level means that the dietary fatcontains less than 10 mg cholesterol per serving of a food containingthe dietary fat, more preferably less than 5 mg per serving, and mostpreferably less than 2 mg per serving to qualify as “cholesterol-free”under current U.S. FDA regulatory standards.

In reference to fatty acids and their attachment to the glyceryl moietyof the triglyceride molecule, there are three hydroxyl positions foresterification of the fatty acids. These positions allow for differenttriglyceride structural isomers, i.e., stereoisomers to be formed. Thethree points of attachment known as the sn-1, sn-2 and sn-3 positionshave metabolic significance. While the physical properties of the fat(e.g., hardness, melting point crystal structure) are affected by eachfatty acid attached at each position, the fatty acid at the middle orsn-2 position has the greatest impact on affecting the level ofdifferent plasma lipoproteins. This is because digestion and enzymatichydrolysis by pancreatic lipase removes the sn-1 and sn3 esterifiedfatty acids, leaving the sn-2 fatty acid monoglyceride to be absorbedinto the bloodstream.

Use herein of the term “fatty acids” refers to such fatty acidsesterified to a glycerol backbone. Primarily the fatty acids will bepresent as triglycerides, although appreciable amounts of di- andmono-glycerides may also be present, along with small amounts of freefatty acids.

As used herein, unless otherwise specified, percentages and theirspecified ranges are provided as weight percentage compositions such as“between 10% and 40% by weight linoleic acid” or from “10% to 40%linoleic acid” Unless clearly indicated to the contrary, all such rangereferences include the endpoints of the range.

Dietary fat compositions as provided and calculated herein are expressedin terms of their fatty acid make-up on a weight percentage basis. Forsimplicity, the total weight percentage of fatty acids intriglyceride-based fats described herein is set to 100% (not ˜95% asused in USDA tables). Thus, the ester-linked glyceryl carbon attached toeach fatty acid is effectively added to that fatty acid because itfacilitates calculations. This concept is described elsewhere herein bythe following alternative words: “the sum of weight percentages forsaturated, polyunsaturated and monounsaturated fat (and fatty acids)equals 100% (based upon the weights of esterified fatty acids in saidcomposition).”

Current methods of chemical and enzymatic interesterification are notdescribed herein because they are well known in the art and aredescribed in the published literature.

The term “unsaturated fatty acids” as used herein refers to fatty acidscontaining at least one carbon-carbon double bond, and as such, includesall fatty acids except the saturated fatty acids. The most commonunsaturated fatty acids include the monounsaturated fatty acid, oleicacid (18:1), and the polyunsaturated fatty acid, linoleic acid (18:2).The polyunsaturates also include the omega-3 fatty acids α-linolenicacid (18:3, n-3 or ALA), and the so-called long chain omega-3polyunsaturated fatty acids, eicosapentaenoic acid (EPA) anddocosahexaenoic acid (DHA). EPA (20:5, n-3) and docosahexaenoic acid(22:6, n-3) contain 5 and 6 double bonds in carbon chains of 20 and 22carbon atoms.

EXAMPLES

Interesterified dietary fats were prepared by the Stepan Company(Northfield, Ill.) using random chemical interesterification to combinethe following fats or fatty acids and vegetable oils:

Example 1

One part by weight trimyristin and three parts by weight high oleicsunflower oil. The sunflower oil (Cargill Inc., Minneapolis, Minn.)contained approximately 82% oleic acid, 8-9% linoleic acid and 8-9%saturated fatty acids. This interesterified fat closely mirrors theinteresterified “olive+14” (75:25) fat of McGandy et al. listed in Table1 (panel 3). Most of the myristic acid in these triglycerides is foundin monomyristin-diolein molecules (liquid oil) whose beta-crystallinemelting point is 14° C., i.e., well below room temperature.

Example 2

Same as Example 1 except 3.9 parts trimyristin and 6.1 parts high oleicsunflower oil are incorporated into the interesterified dietary fat. Theresulting fat contains approximately 5% by weight linoleic acid andapproximately 39% myristic acid, one-third of which (13%) is sn-2myristic acid. Some of the resulting triglycerides will contain twosaturated fatty acids (disaturates) providing a component of fat solidsat room temperature.

Example 3

Same as Example 1 except one part trimyristin and one part high oleicsunflower oil are incorporated into the interesterified dietary fat. Theresulting fat contains only 4% by weight linoleic acid and approximately50% myristic acid, one-third of which is sn-2 myristic acid. If thesunflower oil contains approximately 82% by weight oleic acid (ascurrently available from Cargill Inc., Minneapolis, Minn.), theresulting triglyceride structures with myristic and oleic acids producedby random chemical interesterification/rearrangement include 8 principalstereoisomer structures, 4 of which contain sn-2 myristic acid, i.e.,MMM, MMO, MOM, OMM, MOO, OMO, OOM, OOO. Approximately 40% of themyristic acid resides are found in monomyristin-diolein triglycerideswhile approximately 40% are found in dimyristin-monoolein triglycerides.The remaining myristate (only about 10% of the interesterifiedtriglyceride molecules) is found in trimyristin triglyceride. Thedimyristin-monoolein triglycerides have a convenient beta-primecrystalline melting point of 20-23° C., providing a very useful hard fatfor refrigerated table spreads that will easily melt in ones mouth.

Example 4

Same as Example 3 except one part tripalmitin (instead of trimyristin)and one part high oleic sunflower oil are incorporated into aninteresterified dietary fat. The palmitin-containing interesterified fatproducts can be compared with the myristin-containing homologue productsof Example 3. Used as dietary fats in a controlled nutritional setting,these two products are used to critically test the hypothesis that sn-2myristate-containing triglycerides rather than the homologous sn-2palmitate triglycerides preferentially increase HDL cholesterol andreduce the LDL/HDL cholesterol ratio in human plasma.

As described above, equal amounts of tripalmitin and high oleicsunflower oil are incorporated into an interesterified dietary fat. Theresulting fat contains only 4% by weight linoleic acid and approximately52% palmitic acid, one-third (17%) of which is sn-2 palmitic acid. Ifthe sunflower oil contains approximately 82% by weight oleic acid (ascurrently available from Cargill Inc., Minneapolis, Minn.), theresulting triglyceride structures with palmitic and oleic acids producedby random chemical interesterification/rearrangement include 8 principalstereoisomer structures, 4 of which contain sn-2 palmitic acid, i.e.,PPP, PPO, POP, OPP, POO, OPO, OOP, OOO. Approximately 40% of thepalmitic acid resides are found in monopalmitin-diolein triglycerideswhile approximately 40% are found in dipalmitin-monoolein triglycerides.The remaining palmitin (only about 10% of the interesterifiedtriglyceride molecules) is found in tripalmitin triglyceride. Thedipalmitin-monoolein triglycerides have a beta-prime crystalline meltingpoint of 20-23° C.

Example 5

Same as Example 3 except one part trimyristin and one part regularsafflower oil (Cargill Inc.) are interesterified. The safflower oilprovides a high level of linoleic acid, i.e., 78% by weight, and also13% oleic acid and 9% saturated fatty acids. The result of randominteresterification is much the same as in Example 3 except that thesunflower's oleic acid is replaced by the safflower's linoleic acid (L)to produce principally MMM, MML, MLM, LMM, MLL, LML, LLM, and LLL.

Example 6

A further analysis of the first four exemplary fats described above isprovided below in Table 7, in which these interesterified fats aresubsequently blended, i.e., mixed, with natural safflower oil toincrease the level of linoleic acid in the dietary fat to achieve finallevels of 10%, 15% and 20% by weight linoleic acid.

Dietary Fat Blends with Interesterfied Triglycerides

Ingredients

Trimyristin triglyceride (14:0)

Tripalmitin triglyceride (16:0)

Sunflower oil (hi oleic) [8% SFA (4% 16:0, 4% 18:0), 82% MUFA (18:1), 8%PUFA (18:2)

Safflower oil (regular) [9% SFA (7% 16:0, 2% 18:0), 12% MUFA (18:1), 78%PUFA (18:2)]

Interesterified Fats

IE1: 25% Trimyristin:75% Sunflower (31% SATS, 63% MONOS, 6% POLYS)Triglycerides: mostly monomyristin

IE2: 39% Trimyristin:61% Sunflower (44% SATS, 50% MONOS, 5% POLYS)Triglycerides: intermediate mixture of mono- and dimyristin

IE3: 50% Trimyristin:50% Sunflower (54% SATS, 41% MONOS, 4% POLYS)Triglycerides: approximately 40% monomyristin, 40% dimyristin, 10%trimyristin

IE4: 50% Tripalmitin:50% Sunflower (54% SATS, 41% MONOS, 4% POLYS)Triglycerides: approximately 40% monopalmitin, 40% dipalmitin, 10%tripalmitin

TABLE 7 PERCENTAGES BY WEIGHT BLENDS Myr Sn-2 M Palm SATS Oleic LinoP/S 1. 100% IE1 25 8 3 31 62 6 0.19 2. 94% IE1 + 24 8 3 30 60 10 0.33 6%Saff 3. 87% IE1 + 22 7 4 28 57 15 0.54 13% Saff 4. 80% IE1 + 20 7 4 2753 20 0.74 20% Saff 5. 100% IE2 39 13 2 44 50 5 0.11 6. 93% IE2 + 36 123 42 47 10 0.24 7% Saff 7. 86% IE2 + 34 11 3 39 45 15 0.38 14% Saff 8.79% IE2 + 31 10 3 37 42 20 0.54 21% Saff 9. 100% IE3 50 17 2 54 41 40.07 10. 92% IE3 + 46 15 2 51 39 10 0.20 8% Saff 11. 85% IE3 + 43 14 347.5 37 15 0.32 15% Saff 12. 78% IE3 + 39 13 3 44 35 20 0.45 22% Saff13. 100% IE4 — 17 52 54 41 4 0.07 14. 92% IE4 + — 15 48 51 39 10 0.20 8%Saff 15. 85% IE4 + — 14 46 47.5 37 15 0.32 15% Saff 16. 78% IE4 + — 1342 44 35 20 0.45 22% Saff

Unless otherwise defined herein, all terms have their ordinary meaningsas understood by one of ordinary skill in the field to which theinvention pertains. The use of the article “a” or “an” is intended toinclude one or more.

All patents and other references cited in the specification areindicative of the level of skill of those skilled in the art to whichthe invention pertains, and are incorporated by reference in theirentireties, including any tables and figures, to the same extent as ifeach reference had been incorporated by reference in its entiretyindividually.

One skilled in the art would readily appreciate that the presentinvention is well adapted to obtain the ends and advantages mentioned,as well as those inherent therein. The methods, variances, andcompositions described herein as presently representative of preferredembodiments are exemplary and are not intended as limitations on thescope of the invention. Changes therein and other uses will occur tothose skilled in the art, which are encompassed within the spirit of theinvention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention. Forexample, in addition to the natural, synthetic and semisynthetic dietaryfats listed herein, others that are not listed may be incorporated intothe compositions described herein. Likewise, other sources of myristicacid, sn-2 myristic acid, linoleic acid and other fatty acids and fatsnot listed herein that increase plasma levels of HDL-C, decrease plasmalevels of LDL-C and decrease the ratio of LDL-C/HDL-C, may beincorporated into the compositions described herein, and used incombinations and concentrations not described herein, to producesynthetic and semisynthetic fats that fall within the scope of thepresent invention. Genetically engineered and naturally selected plantspecies that produce fats whose triglycerides are structured and whosefatty acid levels are in accordance with the present invention also fallwithin the scope of the present invention. Thus, such additionalembodiments are within the scope of the present invention and thefollowing claims.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

Also, unless indicated to the contrary, where various numerical valuesor value range endpoints are provided for embodiments, additionalembodiments are described by taking any 2 different values as theendpoints of a range or by taking two different range endpoints fromspecified ranges as the endpoints of an additional range. Such rangesare also within the scope of the described invention. Further,specification of a numerical range including values greater than oneincludes specific description of each integer value within that range.

Thus, additional embodiments are within the scope of the invention andwithin the following claims.

What is claimed is:
 1. An edible fat composition comprising at least 1%by weight myristic acid esterified at the sn-2 position in triglyceridemolecules; between 10% and 40% by weight linoleic acid; between 30% and65% by weight oleic acid; and between 15% and 40% by weight saturatedfatty acids in toto, wherein the weight ratio of sn-2 myristic acid tosn-2 palmitic acid is at least 1:1, the sum of weight percentages forsaturated, monounsaturated and polyunsaturated fatty acids equals 100%;and wherein consistent ingestion of said edible fat increases HDLcholesterol, decreases LDL cholesterol and decreases the LDL/HDLcholesterol ratio in human plasma.
 2. The edible fat composition ofclaim 1 comprising between 5% and 10% by weight myristic acid esterifiedat the sn-2 position in triglyceride molecules.
 3. The edible fatcomposition of claim 1 wherein less than 10% of the triglyceridemolecules comprising myristic acid comprise three myristic acidresidues.
 4. The edible fat composition of claim 1 wherein at least 40%of the triglyceride molecules comprising myristic acid comprise twomyristic acid residues.
 5. The edible fat composition of claim 1 whereinat least 40% of the triglyceride molecules comprising myristic acidcomprise only one myristic acid residue.
 6. The edible fat compositionof claim 1, wherein the percentage by weight of myristic acid esterifiedat the sn-2 glyceride position is between 3% and 5%.
 7. The edible fatcomposition of claim 1, wherein the percentage by weight of myristicacid esterified at the sn-2 glyceride position is between 5% and 8%. 8.The edible fat composition of claim 1, wherein the percentage by weightof myristic acid esterified at the sn-2 glyceride position is between 8%and 12%.
 9. The edible fat composition of claim 1, wherein thepercentage by weight of myristic acid esterified at the sn-2 glycerideposition is between 12% and 16%.
 10. The edible fat composition of claim1, wherein the percentage by weight of myristic acid esterified at thesn-2 glyceride position is between 16% and 20%.
 11. A method ofpreparing a substantially cholesterol-free, edible fat composition,comprising blending a substantially cholesterol-free edible oil rich insn-2 myristate with at least one other substantially cholesterol-freeedible oil, thereby forming a blended edible oil comprising at least 1%by weight myristic acid esterified at the sn-2 position in triglyceridemolecules, between 10% and 40% by weight linoleic acid, between 30% and65% by weight oleic acid, and between 15% and 40% by weight totalsaturated fatty acids, wherein the ratio of sn-2 myristic acid to sn-2palmitic acid is greater than 1:1, and the sum of weight percentages forsaturated, polyunsaturated and monounsaturated fatty acids equals 100%.12. The method of claim 11, wherein said edible oil rich in sn-2myristate is formed by a method comprising enzymatic or chemicalinteresterification.
 13. An edible fat composition comprising at least1% by weight myristic acid esterified at the sn-2 position intriglyceride molecules; between 10% and 40% by weight of at least twopolyunsaturated fatty acids selected from the group consisting oflinoleic acid, alpha-linolenic acid, EPA, DHA, and combinations thereof;between 30% and 65% by weight oleic acid; and between 15% and 40% byweight saturated fatty acids in toto, wherein the weight ratio of sn-2myristic acid to sn-2 palmitic acid is at least 1:1, the sum of weightpercentages for saturated, monounsaturated and polyunsaturated fattyacids equals 100%; and wherein consistent ingestion of said edible fatincreases HDL cholesterol, decreases LDL cholesterol and decreases theLDL/HDL cholesterol ratio in human plasma.